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PHYSIOLOGY 



REVIEW ARTICLE 

published: 24 December 2013 
doi: 10.3389/fphys. 2013. 00386 




Pharmacological rescue of trafficking-impaired 
ATP-sensitive potassium channels 

Gregory M. Martin, Pei-Chun Chen, Prasanna Devaraneni and Show-Ling Shyng* 

Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OB, USA 



Edited by: 

Harley T. Kurata, University of British 
Columbia, Canada 

Reviewed by: 

Mark Dunne, The University of 
Manchester, UK 

Blanche Schwappach, University 
Medicine Gottingen, Germany 
Carol Vandenberg, University of 
California, Santa Barbara, USA 

'Correspondence: 

Show-Ling Shyng, Department of 
Biochemistry and Molecular Biology, 
Oregon Health & Science University, 
L224, 3181 SWSam Jackson Park 
Road, Portland, OR 97239, USA 
e-mail: shyngs@ohsu.edu 



ATP-sensitive potassium (Katp) channels link cell metabolism to membrane excitability 
and are involved in a wide range of physiological processes including hormone secretion, 
control of vascular tone, and protection of cardiac and neuronal cells against ischemic 
injuries. In pancreatic p-cells, Katp channels play a key role in glucose-stimulated insulin 
secretion, and gain or loss of channel function results in neonatal diabetes or congenital 
hyperinsulinism, respectively. The p-cell Katp channel is formed by co-assembly of four 
Kir6.2 inwardly rectifying potassium channel subunits encoded by KCNJ11 and four 
sulfonylurea receptor 1 subunits encoded by ABCC8. Many mutations in ABCC8 or 
KCNJ11 cause loss of channel function, thus, congenital hyperinsulinism by hampering 
channel biogenesis and hence trafficking to the cell surface. The trafficking defects 
caused by a subset of these mutations can be corrected by sulfonylureas, Katp 
channel antagonists that have long been used to treat type 2 diabetes. More recently, 
carbamazepine, an anticonvulsant that is thought to target primarily voltage-gated sodium 
channels has been shown to correct Katp channel trafficking defects. This article reviews 
studies to date aimed at understanding the mechanisms by which mutations impair 
channel biogenesis and trafficking and the mechanisms by which pharmacological ligands 
overcome channel trafficking defects. Insight into channel structure-function relationships 
and therapeutic implications from these studies are discussed. 



Keywords: ATP-sensitive potassium channel, pharmacological chaperone, sulfonylurea, carbamazepine, congenital 
hyperinsulinism (CHI) 



INTRODUCTION 

ATP-sensitive potassium channels (Katp) are a unique class of ion 
channels expressed in a variety of tissues including the pancreas, 
various regions of the brain, and cardiac, skeletal, and vascular 
smooth muscle (Aguilar-Bryan et al, 1998). By regulating K + 
flux at the plasma membrane, they function as molecular sensors 
that couple cell metabolism to changes in membrane excitability 
(Nichols, 2006). The Katp channel is a hetero-octamer formed 
by a complex of two distinct protein subunits in 1:1 stoichiome- 
try: an inwardly rectifying K + channel Kir6. 1/6.2 and a regulatory 
sulfonylurea receptor SUR1 or SUR2 (Inagaki et al, 1995, 1997; 
Clement et al., 1997; Shyng and Nichols, 1997). The "classic" 
channel subtype is composed of a tetramer Kir6.2, which forms 
the K + -conducting pore, with four surrounding SUR1 molecules, 
which provide regulatory functions. 

In pancreatic P-cells, where Katp channels are best stud- 
ied, they act as a key link in glucose-induced insulin secretion 
(Aguilar-Bryan and Bryan, 1999; Ashcroft, 2005). In these cells, 
fluctuations in the [ATP]/[ADP] ratio, brought about by changes 
in blood glucose levels, push the equilibrium of Katp channels 



Abbreviations: CBZ, carbamazepine; CF, cystic fibrosis; CFTR, cystic fibrosis 
transmembrane conductance regulator; CHI, congenital hyperinsulinism; Katp, 
ATP-sensitive potassium channel; Kir6.2, inwardly rectifying potassium channel 
6.2; NBD, nucleotide binding domain; PNDM, permanent neonatal diabetes; SU, 
sulfonylureas; SUR1, sulfonylurea receptor 1; TMD, transmembrane domain. 



toward the closed or open state. Thus, when blood glucose lev- 
els rise, the intracellular [ATP]/[ADP] ratio also rises, blocking 
K + efflux through Katp channels. This depolarizes the P-cell and 
opens voltage-gated Ca 2+ channels; the subsequent Ca 2+ influx 
then triggers exocytosis of insulin secretory granules. When blood 
glucose levels fall, the intracellular [ATP]/[ADP] ratio will fall, 
pushing the equilibrium toward open Katp channels, repolarizing 
the P-cell and blocking further insulin release (Figure 1). 

Not surprisingly, mutations in the genes encoding Katp chan- 
nel subunits (ABCC8 for SUM and KCNJ11 for Kir6.2) often 
lead to a breakdown in glucose homeostasis. In general, muta- 
tions in Katp genes are classified as either gain-of-function, 
where constitutively open channels preclude insulin secretion, 
or loss-of-function, non-functional channels that are unable 
to hyperpolarize the p-cell and cause persistent insulin release 
(Aguilar-Bryan and Bryan, 1999; Hattersley and Ashcroft, 2005). 
Over the past 15 years, a number of groups have identified a 
class of loss-of-function mutations in the genes encoding the 
Katp channel, particularly in ABCC8 (SUM), that interfere with 
proper biogenesis and trafficking, thus, preventing normal sur- 
face expression of the channel (Cartier et al, 2001; Partridge et al., 
2001; Taschenberger et al., 2002; Crane and Aguilar-Bryan, 2004; 
Tornovsky et al, 2004; Yan et al, 2004, 2007; Taneja et al, 2009). 
These mutations are collectively referred to as trafficking muta- 
tions. Studies have demonstrated that congenital hyperinsulinism 
of infancy (CHI), a rare disease characterized by persistent insulin 



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Martin et al. 



Correcting Katp channel trafficking defects 



Resting state 

Voltage-gated 




FIGURE 1 | K A tp channels regulate insulin secretion in pancreatic p-cells. 

Under conditions of low blood glucose, the relatively low ATP/ADP ratio in 
the p-cell promotes opening of K ATP channels, keeping p-cell membrane 
potential at a hyperpolarized state to prevent Ca 2+ influx and insulin release. 
Upon an increase in blood glucose, p-cells increase glucose uptake through 



secretion even under severe hypoglycemia (Stanley, 1997), is fre- 
quently caused by trafficking mutations in Katp channel genes. In 
these patients, channel subunits are synthesized but fail to reach 
the plasma membrane, mostly due to a disruption in the fold- 
ing or oligomeric assembly process. The result is a constitutively 
depolarized fi-cell with unregulated levels of insulin release. In 
many cases, the current therapy for these patients relies on par- 
tial or subtotal pancreatectomy to avoid permanent consequences 
of chronic hypoglycemia, which could lead to life-long insulin 
dependency. 

Protein misfolding and mistrafficking resulting from genetic 
mutations underlie many human diseases. A prominent example 
is the AF508-CFTR (cystic fibrosis transmembrane conductance 
regulator) deletion mutation (Riordan et al., 1989), which is 
present in the majority of cystic fibrosis (CF) patients and causes 
defective folding, thereby inhibiting trafficking of the protein to 
the plasma membrane (Cheng et al., 1990). Small-molecule cor- 
rectors, termed pharmacological chaperones, which specifically 
bind to a protein and enable its proper folding and localization, 
have been shown to correct trafficking defects in multiple disease- 
causing proteins, like AF508-CFTR (Hanrahan et al., 2013). In 
some cases, mutant proteins rescued to the correct cellular loca- 
tions exhibit full or partial function to reverse disease phenotypes 
(Powers et al., 2009). Recent work has demonstrated that phar- 
macological chaperones may also hold promise in correcting 
trafficking-impaired Katp channels, offering new hope in the 
treatment of CHI. 

In this review, we will discuss progress to date in defining the 
mechanisms by which mutations impair the biogenesis and traf- 
ficking of Katp channels and how these trafficking defects can 
be overcome using pharmacological approaches. In particular, 
we will describe the challenges facing pharmacological rescue of 
trafficking-impaired ion channels, and discuss the promises this 
area holds in the treatment of disease. 



Glucose-stimulated state 

Voltage-gated 




GLUT2 transporters; glycolysis and respiration then elevate the intracellular 
ATP/ADP ratio and close Katp channels. This causes depolarization of the 
plasma membrane potential which opens voltage-gated Ca 2+ channels; the 
subsequent influx of Ca 2+ initiates fusion of insulin secretory granules with 
the plasma membrane. 



MOLECULAR COMPOSITION OF K ATP CHANNELS 

The Katp channel is a large hetero-octamer of nearly 950 kDa, 
composed of four Kir6.2 and four SUR1 subunits (Clement et al., 
1997) (Figure 2). A low-resolution cryo-EM structure indicates a 
compact configuration, 18 nm across and 13 nm in height, with 
a central tetrameric Kir6.2 core which forms the K + -conducting 
pore, embraced by four SUR1 proteins (Mikhailov et al., 2005). 

Kir6.2 is a member of the potassium inward rectifier family 
(Kir). These channels have a greater tendency to allow ion flow 
into, rather than out of the cell owing to block by intracellular 
polyamines and Mg 2+ at positive membrane potentials (Lopatin 
et al., 1994). In the case of weak inward rectifiers such as Kir6.2, 
the extent of intracellular block is less pronounced due to lack 
of strong binding sites for the positively charged blockers. Under 
most physiological conditions where membrane potential is posi- 
tive to the equilibrium potential of K + (Ek), Kir channels generate 
small outward currents to keep membrane potential near the 
Er. This makes Kir channels primary regulators of resting mem- 
brane potential in cells that express them. Importantly, many Kir 
channels are ligand-gated, endowing them the ability to couple 
specific physiological signals to membrane excitability (Nichols 
and Lopatin, 1997). For the Kir6.2-SUR1 Katp channel com- 
plex, gating regulation by intracellular nucleotides ATP and ADP 
underlies its physiological function of coupling cell metabolism 
to cell excitability. 

No definitive high resolution structure for Kir6 channels yet 
exists. However, homology modeling using crystal structures of 
eukaryotic and prokaryotic Kir channels has provided the basis 
for a structural model (Capener et al., 2000; Loussouarn et al., 
2001). Thus, four Kir6.2 subunits combine to form the K + pore. 
Each subunit has two transmembrane domains, Ml and M2, with 
intracellular N- and C-termini. Lying perpendicular to Ml and 
M2 is the short amphipathic "slide helix," which links Ml to the 
short N-terminal domain. Mutagenesis studies have implicated 



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Martin et al. 



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SUR1 

TMDO TMD1 



Kir6.2 

























M1 




M2 












-S1238 I 
(A Site) | 







f NBD2J 



Stimulatory: MgADP 
Diazoxide 



Inhibitory: Sulfonylureas, glinides, 



PIPs, LCacylCoA 
ATP 



FIGURE 2 | Molecular composition and regulation of K ATP channels. 

Pancreatic Katp channels are hetero-octamers of four Kir6.2 subunits, 
which form the K+ conducting pore, and four regulatory SUR1 subunits. 
Shown on the top are transmembrane topologies of the two subunits. 
SUR1 has three transmembrane domains, TMDO, TMD1, and TMD2, two 
cytoplasmic nucleotide binding domains, NBD1 and NBD2, and a 
cytoplasmic linker LO that connects TMDO to the ABC core structure of the 
protein. Kir6.2 has two transmembrane helices, M1 and M2, and 
cytoplasmic N-and C-terminus. Physiological and pharmacological ligands 
that impact channel function are shown below. Mg-nucleotides interact 
with NBDs of SUR1 to activate the channel, whereas ATP binding at Kir6.2 
closes the channel. PIP2 and LC acyl CoAs also interact with Kir6.2 but 
stimulate channel activity. Sulfonylureas and glinides inhibit, whereas 
diazoxide stimulates channel activity by interacting with primarily SUR1. For 
detailed discussion on the involvement of each subunit in channel 
regulation by the various physiological and pharmacological ligands please 
refer to the main text. Locations of the RKR motifs in SUR1 and Kir6.2 are 
marked. Y230 and S1238, SUR1 residues critical for site B and site A of the 
glibenclamide binding pocket are also marked. 



this region in channel gating (Proks et al., 2004; Lin et al., 2006b; 
Li et al., 2013). Interspersing Ml and M2 is an extracellular linker 
followed by a pore-forming loop, constituting the selectivity filter, 
the region responsible for K + ion specificity. The M2 helix con- 
nects to the large intracellular C-domain. This region constitutes 
nearly half of the protein, and mediates extensive interactions 
with adjacent Kir6.2 subunits (Antcliff et al., 2005). 

SUR1 is a member of the ATP Binding Cassette (ABC)-C 
family of transporters, and is closely related to CFTR and MRP 
(multi-drug resistance related proteins). Homology modeling has 
been challenging without a solved structure for an ABC pro- 
tein with significant sequence identity. Nonetheless, biochemical, 
electrophysiological, and mutagenesis studies have provided the 
essential topology and domain organization (Aguilar-Bryan et al., 
1995; Tusnady et al, 1997; Conti et al, 2001) (Figure 2). As an 
ABC transporter, SUR1 has the core ABC structure of two trans- 
membrane domains (TMD1 and TMD2), each consisting of 6 
transmembrane helices. Each TMD is linked to a cytoplasmic 
nucleotide-binding domain (NBD1 and NBD2) by intracellu- 
lar coupling domains (ICDs), a-helical extensions of the TMDs. 
Additionally, SUR proteins contain an N-terminal TMDO domain 
of five transmembrane helices, plus a long cytoplasmic loop L0, 
linking TMDO with the core ABC structure. TMDO is absent 
from most ABC transporters, including CFTR, and seems to play 
a unique and interesting role in Katp channel biology. Studies 



implicate TMDO in mediating interactions between SUR1 and 
Kir6.2 and modulating forward trafficking and gating of the chan- 
nel (Schwappach et al., 2000; Babenko and Bryan, 2003; Chan 
et al., 2003). Another interesting point regarding SUR1 is the fact 
that this ABC-C transporter has no known function as a trans- 
porter; its role is strictly regulatory with regard to Kir6.2, at least 
in the systems examined so far. How these two proteins evolved 
to form a functional channel complex is a fascinating question. 

BIOGENESIS AND TRAFFICKING REGULATION OF K ATP 
CHANNELS 

Biogenesis and assembly of Katp channel proteins occurs in 
the ER (Zerangue et al., 1999). Not much is known regarding 
the events that couple translation of Katp channel subunits to 
insertion in the ER membrane however, or regarding details of 
the assembly process and molecular chaperones involved. When 
either SUR1 or Kir6.2 is expressed alone in heterologous systems, 
the subunits cannot escape the ER (Zerangue et al, 1999) and are 
presumably cleared through ER-associated degradation (ERAD), 
suggesting that assembled complexes are required for forward 
trafficking and surface expression. Yan et al. confirmed that ubiq- 
uitin and proteasome-mediated ERAD is a primary check on 
Katp channels during biogenesis and that this process in part 
regulates the surface expression of Katp channels as inhibition 
of proteasome function led to an increase in surface expression 
of the channel (Yan et al., 2005). More recently, Wang and col- 
leagues showed that Derlin-1, an ER membrane protein involved 
in recognition or retrotranslocation of substrates out of the ER for 
ERAD (Lilley and Ploegh, 2004; Ye et al, 2004), forms a complex 
with SUR1 and Kir6.2 and is also an important factor determining 
surface levels of Katp channels (Wang et al., 2012). SUR1 is a gly- 
coprotein, containing two N-linked glycosylation sites. Conti et al. 
mutated these sites in SUR1 and observed ER retention of the 
protein, suggesting that lectin chaperones calnexin/calreticulin, 
which are known to assist the folding of glycoproteins, participate 
in the folding and assembly of Katp channels (Conti et al., 2002). 
Yan et al. also demonstrated that the molecular chaperone Hsp90 
participates in the folding of Katp channels by interacting with 
SUR1, and that knockdown of Hsp90 reduced surface expres- 
sion of the channel (Yan et al., 2010). These studies only begin 
to address the sequence of events and the mechanisms that gov- 
ern the folding/assembly and degradation of the Katp channel, 
and represent important first steps in understanding this critical 
aspect of Katp channel biology. 

ASSEMBLY DOMAINS 

A central question in understanding channel biogenesis is what 
are the assembly domains on the subunits themselves that direct 
this process? Using chimeras of Kir2.1, which is known to not 
associate with SUR1, and Kir6.2, Giblin et al. looked for mini- 
mal domains necessary for interactions between Kir6.2 and SUR1 
(Giblin et al., 1999). They found that a proximal region (amino 
acids 208-279) in the Kir6.2 C-terminus is necessary for co- 
immunoprecipitation of the two subunits but this region is not 
sufficient to mediate formation of Katp functional channel com- 
plexes as no channel activity could be detected at the cell surface. 
Schwappach et al., with a similar approach using chimeras of 



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Martin et al. 



Correcting Katp channel trafficking defects 



Kir6.2 and Kir2.1 demonstrated the Ml and N-terminus of Kir6.2 
are involved in Katp channel assembly and gating (Schwappach 
etal., 2000). 

One particularly interesting assembly domain of the Katp 
channel is TMDO, the first bundle of transmembrane helices 
in SUR1. This domain, coupled with the first intracellular loop 
L0, is largely unique to SUR proteins; most eukaryotic ABC 
transporters have only the core structure of TMD1 and TMD2 
with the two intracellular nucleotide binding domains NBD1 
and NBD2 (Tusnady et al, 1997). Using chimeric SUR1 pro- 
teins containing TMDO from another ABC transporter MRP1 
known to not interact with Kir6.2, Schwappach et al. first demon- 
strated a role of TMDO in mediating subunit interactions between 
Kir6.2 and SUR1 and in promoting forward trafficking of Kir6.2 
(Schwappach et al, 2000). Subsequent work by others showed 
that in truncated Kir6.2 subunits (see below), TMDO alone 
will increase surface expression of Kir6.2 and produce so-called 
"mini-KATp" channels that display single-channel kinetics sim- 
ilar to WT channels, but are unresponsive to metabolic signals 
and pharmacological ligands (Babenko and Bryan, 2003; Chan 
et al, 2003). Studies utilizing naturally occurring TMDO muta- 
tions present in patients with CHI have identified residues which 
may be crucial for folding or subunit interactions (Chan et al., 
2003; Yan et al, 2004, 2007; Pratt et al, 2011), but the pre- 
cise nature of the interface of TMDO with Kir6.2 remains to be 
elucidated. 

EXITING THE ER 

As mentioned, forward trafficking and surface expression of Katp 
channels relies on assembly of both subunits in the ER. This 
makes Kir6.2 unique among Kir channels, thereby prompting 
studies to define the domain responsible for ER retention of 
unassembled Kir6.2 subunits. Tucker et al. first demonstrated 
that C-terminal truncations of 25 or 36 amino acids in Kir6.2 
allowed for potassium currents in the absence of SUR1 (Tucker 
et al., 1997). Later work by Zerangue et al. utilized this semi- 
nal discovery in mapping the particular retention motif within 
the C-terminus of Kir6.2 and identified a tripeptide RKR local- 
ization signal (Zerangue et al., 1999) which was found to also 
function in SUR1 (Figure 2). The current model suggests these 
motifs must be masked during the assembly of the channel com- 
plex to allow forward trafficking to proceed. Unassembled or 
misassembled subunits are thus prevented from reaching the 
plasma membrane, allowing for quality control in the Katp bio- 
genesis pathway. Questions still remain regarding the mechanism 
of how the RKR motifs function in ER retention. A study by 
Yuan et al using an artificial reporter construct first brought 
forth a model whereby an interplay between the 14-3-3 fam- 
ily of proteins and the coatamer complex 1 (COPI) acts to 
regulate ER to Golgi trafficking via interactions with the RKR 
motifs (Yuan et al., 2003). Their group showed that COPI pro- 
teins can specifically bind the RKR motif in Kir6.2. The same 
study demonstrated 14-3-3 proteins can also recognize the sig- 
nal when multiple subunits are present. The model proposes 
that COPI proteins recognize RKR motifs on misassembled or 
unassembled subunits and promote their retrieval to the ER. 
The 14-3-3 proteins, by contrast, act as sensors for assembled 



subunits, and can bind properly assembled complexes and pre- 
vent recognition of their RKR motifs by COPI, allowing forward 
trafficking to proceed. A later study from the same lab using the 
14-3-3 scavenger approach further substantiated a role of 14-3-3 
proteins in regulating trafficking and surface expression of het- 
erologously and endogenously expressed Katp channels (Heusser 
et al, 2006). 

Anterograde, or forward trafficking signals on Katp channel 
subunits have been more challenging to define. While TMDO 
clearly facilitates Kir6.2 expression, it can only do so when 
co-expressed with Kir6.2AC26, a C-terminal deletion construct 
missing the last 26 amino acids, in which the RKR motif resides 
(Chan et al, 2003). TMDO, therefore, is not the domain shield- 
ing the RKR signals from COPI proteins. Sharma et al. (1999) 
identified a putative forward trafficking signal in the distal C- 
terminus of SUR1 by making SUR1 constructs missing varying 
numbers of residues in the C-terminus (Sharma et al., 1999). 
However, another study showed that larger deletions in the SUR1 
C-terminus had no effect on channel expression, making the 
existence of the C-terminal forward trafficking signal debatable 
(Giblin et al., 2002). No studies to date have resolved this issue or 
confirmed the existence of a bona fide anterograde signal on either 
subunit. 

Interestingly, studies examining the kinetics of the biogene- 
sis pathway using metabolic pulse-chase labeling have demon- 
strated the intrinsic inefficiency of Katp assembly, estimating that 
only about 20% of newly synthesized SUR1 or Kir6.2 actually 
forms mature complexes, with the remaining pool being rapidly 
degraded (Yan et al, 2004, 2005, 2010; Chen et al, 2011). CFTR 
and other ABC transporters also display a similar level of effi- 
ciency (Ward and Kopito, 1994). Yan et al. have shown that both 
SUR1 and Kir6.2 exhibit a biphasic degradation profile when 
expressed alone, each containing a fast and a slow component 
(Yan et al., 2004, 2005). Crane and Aguilar-Bryan also observed 
biphasic degradation of Kir6.2 expressed alone; however, they 
reported remarkable stability of SUR1 expressed alone (~25h) 
(Crane and Aguilar-Bryan, 2004). These differences may be due 
to technical reasons or experimental conditions. Nevertheless, 
both groups saw increased stability of SUR1 and Kir6.2 when the 
two subunits were co-expressed, suggesting the subunits become 
more stable upon mulitimeric assembly (Crane and Aguilar- 
Bryan, 2004; Yan et al., 2005). While some differences are yet 
to be resolved, studies like these address fundamental aspects of 
Katp channel biology not by looking at snapshots, but by getting 
the dynamics involved in the biogenesis pathway. Many questions 
remain unanswered: How are nascent polypeptides recognized in 
the cytosol and translocated in the ER? What is the sequence of 
events involving folding and assembly of Katp channel subunits? 
Is folding co- or post-translational? What protein-protein interac- 
tions occur in the ER that guide this process, and what molecular 
chaperones are involved? What routes do Katp channels take after 
exiting the ER? Obviously much work needs to be done, but 
answers to these questions will provide a deep level of insight 
into how large, multimeric membrane proteins are assembled, 
and importantly, will allow researchers to more fully understand 
mechanisms of proteostasis and trafficking diseases, like CHI 
or CF. 



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Martin et al. 



Correcting Katp channel trafficking defects 



GATING REGULATION OF K ATP CHANNELS 

It is well-understood that the primary mode of physiological 
regulation of Katp channel function is through the opposing 
inhibitory action of ATP and stimulatory action of MgADP. The 
bulk ATP concentration is relatively stable in most cells, how- 
ever, raising doubt that ATP can serve as a primary regulator 
of the channel. In f^-cells, ATP concentrations have been esti- 
mated to change from 2 to 4 mM when glucose is elevated from 
0 to 10 mM (Detimary et al., 1998); such high levels of ATP are 
expected to prevent channel opening even at the lowest glucose 
concentrations. However, the small changes in [ATP], coupled 
with much larger changes in ADP levels in the opposite direction 
as glucose concentrations fluctuate, result in significant changes 
in the [ATP]/[ADP] ratio (Nilsson et al, 1996; Detimary et al, 
1998) to shift the apparent ATP sensitivity and effectively regu- 
late channel activity (Tarasov et al, 2004). Thus, when glucose 
levels are low, MgADP stimulation will predominate, Katp chan- 
nels will be open, the cell will be hyperpolarized, and no insulin 
will be secreted. When glucose levels are high, ATP inhibition will 
take over, closing Katp channels, causing membrane depolariza- 
tion, opening voltage-gated Ca 2+ channels and triggering insulin 
exocytosis. 

In the absence of ATP, Katp channels display so-called burst 
kinetics, characterized by periods of rapid openings and closings 
separated by long closed intervals (Alekseev et al., 1997). ATP 
inhibition of Katp channels, involving non-hydrolytic binding 
at the intracellular face of Kir6.2, acts to decrease the frequency 
and length of the burst periods, and increases the duration of 
the closed intervals (Babenko et al, 1999). Based on mutagenesis 
and docking studies using homology models of Kir6.2, binding is 
thought to occur at an interface involving the N- and C-domains 
of one subunit, and the N-domain of an adjacent Kir6.2 subunit 
(Antcliff et al., 2005). Each channel contains four ATP-binding 
sites, yet ATP binding at a single site is sufficient to close the chan- 
nel, in support of a concerted gating model (Markworth et al., 
2000; Drain et al., 2004). Interestingly, however, SUR1 sensitizes 
Kir6.2 to ATP inhibition by about a factor of 10 (Tucker et al., 
1997). This could be due to allosteric effects on Kir6.2 or facilita- 
tion of ATP binding by SUR1 (Babenko, 2005); at this point the 
mechanism remains unclear. 

Nucleotide activation occurs at the nucleotide binding 
domains of SUR1 and acts to antagonize ATP inhibition (Gribble 
et al., 1997). The requirement of Mg 2+ in this process has 
prompted studies to examine nucleotide interactions and hydrol- 
ysis at the NBDs. These studies led to a proposal that hydrolysis 
of MgATP to MgADP at NBD2, which contains the consensus 
ATPase site, stabilizes ATP binding at NBD1, which carries a 
degenerate ATPase site, and drives dimerization of the NBDs to 
promote channel opening at Kir6.2 (Zingman et al, 2001; Matsuo 
et al, 2005; Masia and Nichols, 2008). It is worth noting that 
direct measurements of MgATP hydrolysis using purified SUR or 
recombinant SUR NBDs indicate relatively poor hydrolysis effi- 
ciency (Masia et al., 2005; De Wet et al., 2007), suggesting that 
increased MgADP binding at NBD2 as intracellular [ADP] rises 
may be sufficient to induce or stabilize conformational changes at 
the NBDs to stimulate channel opening. Recent work by Ortiz 
et al. using glibenclamide binding to probe switching of the 



NBDs between closed and open dimer conformations supports 
the notion that nucleotide hydrolysis at NBD2 is not required for 
conformational switch (Ortiz et al., 2012, 2013). Physiologically, 
the regulation of SUR1 in response to an increase in intracellu- 
lar [ADP] is critical for repolarizing the fi-cell when glucose levels 
drop; without it the fi-cell will be unable to stop secreting insulin. 
This is one common mechanism for mutations identified in SUR1 
in patients with CHI (Nichols et al, 1996a; Shyng et al, 1998), 
who persistently secrete insulin even under severely low blood 
glucose levels. 

Membrane phosphoinositides, such as phosphatidylinositol- 
4,5-bisphosphate (PIP2), and metabolic derivatives of free fatty 
acids, long-chain acyl-CoA esters (LC-CoAs) also have pro- 
found effects on Katp channel gating (Baukrowitz et al., 1998; 
Branstrom et al, 1998; Shyng and Nichols, 1998). Both PIP 2 and 
LC-CoAs interact with Kir6.2 via similar mechanisms to increase 
channel open probability and antagonize ATP inhibition (Schulze 
et al, 2003). Mutagenesis studies indicate that PIP2 and ATP may 
have overlapping but non-identical binding sites in Kir6.2 (Shyng 
et al, 2000; Cukras et al, 2002a,b; Antcliff et al, 2005). While 
Kir6.2 interacts directly with PIP2, SUR1 increases the sensitiv- 
ity of the channel to PIP2 stimulation (Baukrowitz et al., 1998; 
Shyng and Nichols, 1998). Recent work by Pratt et al. shows that 
TMD0 of SUR1, which is known to increase the open probabil- 
ity of Kir6.2, does so by stabilizing the interaction between Kir6.2 
and PIP 2 (Pratt et al., 2011). A crucial role of PIP2 in maintain- 
ing channel activity in P-cells has been clearly demonstrated (Lin 
et al, 2005); mutations which disrupt the channel response to 
PIP2 have been identified in patients with CHI (Lin et al., 2006b, 
2008). The relevance of PIP20r LC-CoAs to physiological regula- 
tion of Katp activity however, is less certain (Tarasov et al., 2004), 
although pathological changes in LC-CoAs have been proposed to 
impact channel function (Larsson et al., 1996; Riedel and Light, 
2005). 

PHARMACOLOGICAL REGULATION OF K ATP CHANNELS 

An important class of agents which regulate Katp channels 
are sulfonylureas (Aguilar-Bryan and Bryan, 1999; Gribble and 
Reimann, 2003b). Discovered in the 1940s as sulfonamide drugs 
that have hypoglycemic effects, sulfonylureas interact specifi- 
cally with the sulfonylurea receptor SUR, giving it its namesake. 
Sulfonylureas have been used in the treatment of non-insulin 
dependent diabetes for decades, yet precisely how they promote 
insulin release in the body remained a mystery for years. It is 
now well understood that sulfonylureas bind directly to SUR1 and 
inhibit channel activity, thus stimulating insulin release by depo- 
larizing the P-cell independent of glucose. Tolbutamide was part 
of the first generation of hypoglycemic compounds, later replaced 
by second-generation compounds like glibenclamide, which have 
100 to 1000-fold more potency in blocking Katp currents. A 
related group of sulfonamide compounds, most notably diazox- 
ide, also bind specifically to SUR1 but activate channels (Dunne 
et al., 1989; Moreau et al., 2000). Diazoxide is part of a diverse 
group of drugs that stimulate K + currents, known as potassium 
channel openers (KCOs) (Manley et al., 1993). 

Although the Katp channel was first identified in cardiac 
myocytes by Aki Noma in 1983 (Noma, 1983), its molecular 



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Martin et al. 



Correcting Katp channel trafficking defects 



identify remained uncertain for many years. Sulfonyulureas, 
owing to their specificity, were used as probes in the purifica- 
tion and cloning of Katp channels. Early patch-clamp experi- 
ments demonstrated that sulfonylureas were specific blockers of 
|3-cell Katp channels, while diazoxide specifically opened them. 
A significant advancement came from using labeled derivatives 
of glibenclamide, [ 3 H]glibenclamide (Kramer et al., 1988), and 
later [ 125 I]iodoglibenclamide (Aguilar-Bryan et al., 1990), which 
could label a 140-kDa protein (SUR1) in isolated membranes 
of various fi-cell lines and led to cloning of the SUR1 gene 
(Aguilar-Bryan et al, 1995). Schwanstecher et al. further used 
125 I-iodo-azidoglibenclamide, an azido analog of glibenclamide, 
and found it would co-photolabel a 38 kDa protein in addition 
to SUR1 and with the same apparent Kd (Schwanstecher et al., 

1994) ; this species was later identified as Kir6.2 (Inagaki et al., 

1995) . This provided the first evidence that the Katp channel is a 
multimeric complex of Kir6.2 and SUR1. 

Significant progress has since been made in defining the pre- 
cise mechanism by which sulfonylureas and glinides, another 
class of Katp channel inhibitors used in diabetes therapy, block 
Katp channel function. All sulfonylureas and glinides demon- 
strate high and low-affinity components of inhibition, such that 



A B-site A-site 



their dose-response curves are generally biphasic, with variable 
separation between the two sites (Gribble and Reimann, 2003a). 
At low concentrations, interaction with the SUR subunit results 
in 50-75% reduction in current amplitude when administered to 
the cytoplasmic face of membrane patches, suggesting that chan- 
nels can remain open to an extent when bound to the drug. The 
low-affinity component is attributed to interaction with Kir6.2, 
but this only occurs at very high drug concentrations. 

According to the pharmacophore model, structurally diverse 
compounds can possess overlapping electronic and stereochem- 
ical properties that allow them to bind a common receptor 
site. SUR was proposed to bind sulfonylureas accordingly with 
a bipartite binding pocket (Figure 3A). Consistent with this, 
the enhanced affinity observed in glibenclamide is attributed to 
interaction with the two overlapping binding sites on SUR1, 
termed site A and site B (Brown and Foubister, 1984). Site A 
is proposed to interact with a lipophilic group adjacent to the 
negative charge of a sulfonylurea group, while site B recognizes 
a lipophilic group adjacent to an amide. Glibenclamide pos- 
sesses both of these moieties, while most other sulfonylureas 
and hypoglycemic agents such as glinides possess one or the 
other. Sulfonylureas are further distinguished by their ability 



B 




Carbamazepine 




Tolbutamide 



Glibenclamide 



Repaglinide 



F27S 



A116P 



^^0.2 



CBZ (uM) 



6. 5? 



CBZ (nM) 



1 10 50 -f 0.2 



V187D 
CBZ (uM)" 



0.2 1 10 50 



Tubulin |— - — » — — — — 1 



"1-250 
1 — 150 



■ 50 



FIGURE 3 | The processing defect of SUR1 caused by TMD0 trafficking 
mutations is corrected by glibenclamide and carbamazepine. (A) 

Pharmacophore model for binding of various K ATP channel blockers that 
function as effective pharmacological chaperones, showing the chemical 
moieties thought to confer affinity for either site A or site B on SUR1. 
(B) Chemical structure of carbamazepine. (C) Western blot of whole cell 
lysates of COSm6 cells transiently transfected with Kir6.2 plus WT or TMD0 
mutant SUR1 cDNAs. SUR1, a glycoprotein, shows two bands in 



immunoblots: a lower core glycosylated (immature; open arrow) form that 
has not exited the ER and an upper complex-glycosylated (mature; solid 
arrow) band that has trafficked through the Golgi. Incubation of cells with 
glibenclamide (5 (iM; Glib) or carbamazepine (CBZ) for 16 h increased levels 
of the mature band of TMD0 trafficking mutants compared to those treated 
with DMSO (veh). The effect of carbamazepine was dose-dependent, with an 
effect detectable at a concentration as low as 0.2 \lM. Untransfected control 
is shown for comparison. Adapted from Figure 1 in Chen et al. (2013b). 



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Martin et al. 



Correcting Katp channel trafficking defects 



to block either SUR1 or SUR2 Katp channels. Compounds 
that contain a non-sulfonylurea moiety (such as glibenclamide, 
glimepiride, repaglinide) can bind with high affinity to SUR1 
and SUR2, while those with only the core sulfonylurea struc- 
ture (tolbutamide, gliclizide) are specific for SUR1 (Quast et al., 
2004). This difference made tolbutamide a pharmacological tool 
to uncover the sulfonylurea binding (A) site. Research using 
chimeric receptors of SUR1/SUR2 showed that TMD2 of SUR1 
is critical for sulfonylurea binding. Further studies showed that 
mutation of SI 23 7 of the cytoplasmic loop between TMs 15 
and 16 to tyrosine (the equivalent residue in SUR2) abolished 
high affinity tolbutamide block and [ 3 H] glibenclamide bind- 
ing (Ashfield et al, 1999) [note that S1237 in SUR1 is now 
referred to as S1238, as current sequence information for ABCC8 
(GenBank reference NM_00352.2) includes the alternate exon 17 
(GenBank L78208) which contains an additional amino acid]. 
Further, the reciprocal mutation in SUR2 (Y1206S) increased 
[ 3 H] glibenclamide binding by roughly 10-fold. Mapping of site 
B in SUR1 came from a study examining affinity labeling with 
[ 125 I]iodoglibenclamde which identified a ~50kDa fragment 
including TMD0-L0 (Bryan et al., 2004). A more precise defini- 
tion came from Vila-Carriles et al. by using deletions and alanine 
scanning in TMDO and L0 of SUR1 to monitor photolabeling 
by [ 125 I]azido-iodoglibenclamide (Vila-Carriles et al., 2007). The 
authors concluded that L0 of SUR1 is absolutely essential for 
glibenclamide binding, while TMDO is nonessential. Scanning 
alanine mutations showed that Y230 and W232 are critical for 
high affinity photolabeling. Further, they determined that the N- 
terminal 33 amino acids of Kir6.2 are involved in site B labeling 
by [ 125 I]azido-iodoglibenclamide. Together, these results indicate 
that site A (TMD2) and site B (L0) are within close physical prox- 
imity such that they can cooperatively bind glibenclamide, and 
that the N-terminus of Kir6.2 also forms part of the site B binding 
pocket. 

PHARMACOLOGICAL REGULATION OF K ATP CHANNEL 
TRAFFICKING DEFECTS 

Over the past 10 years, it has been recognized that, in addition 
to their action as specific Katp channel blockers, sulfonylureas 
also promote the proper folding and biogenesis of trafficking- 
impaired mutant SUR1 proteins identified in patients with CHI 
by acting as pharmacological chaperones. They achieve this with 
domain specificity, only rescuing mutations within TMDO of 
SUR1. In principle, this opened up a new therapeutic avenue for 
sulfonylureas, in which they could be administered to patients 
with TMDO trafficking mutations and rescue Katp channel func- 
tion in P-cells. Sulfonylureas are imperfect correctors, however, as 
they often irreversibly block channel function of rescued mutant 
channels. This is an unsuitable therapeutic approach for treating 
diseases like CHI, which require functional channels at the cell 
surface in order to limit insulin release and restore blood glucose 
to normal levels. Recently, additional new compounds such as 
carbamazepine (CBZ) (Figure 3B), which also correct trafficking- 
impaired Katp channels with mutations in TMDO of SUR1 with- 
out the irreversible block observed with glibenclamide, have been 
identified. As discussed below, in vitro studies with pharmacolog- 
ical chaperones have increased our understanding of how certain 



Katp channel mutations lead to disease, strengthening the link 
between genotype and phenotype, while also highlighting gen- 
eral principles involved in the gating and molecular assembly of 
heteromeric ion channels, like Katp. 

K AT p CHANNEL TRAFFICKING DEFECTS IN HUMAN DISEASE 

Proper cellular function relies on both the absolute number and 
subcellular localization of many proteins; this is particularly true 
of membrane proteins like Katp- For ion channels and other 
membrane or secreted proteins, translation begins in the cytosol 
but soon becomes associated with the endoplasmic reticulum 
(ER). Here it is thought the bulk of protein folding and qua- 
ternary assembly take place. This occurs under the auspices of 
an array of molecular chaperones, integral members of the ER 
quality control system, which ensure the proper folding, process- 
ing, and structural integrity of nascent proteins while preventing 
the accumulation of defective proteins which may disrupt normal 
cell function. Trafficking mutations can interfere with this process 
by disrupting protein folding and molecular assembly in the ER, 
generally leading to retention and clearance of mutant proteins 
through ERAD. 

Mislocalization of cell-surface proteins which are otherwise 
functional has been demonstrated in numerous diseases, includ- 
ing CF, familial hypercholesteremia, retinitis pigmentosa, and 
diabetes insipidus (Powers et al., 2009). In CF, the AF508 deletion 
(present in 90% of CF patients) in CFTR leads to ER retention and 
rapid degradation of the incompletely processed protein by the 
proteasome (Jensen et al, 1995; Ward et al., 1995). Insufficient 
levels of this chloride channel at the cell surface prevent cAMP- 
mediated chloride ion, water, and bicarbonate conductance in 
a variety of tissues. More recently, it has been established that 
point mutations in the genes encoding the Katp channel sub- 
units can also interfere with or prevent proper folding and/or 
molecular assembly in the ER (Table 1). These mutations are 
found throughout the SUR1 and Kir6.2 proteins. Normal lev- 
els of protein are usually translated, but mutant proteins are 
unable to exit the ER, and as with AF508-CFTR and many other 
conformationally-defective proteins, are degraded through the 
ubiquitin/proteasome pathway. Katp channel trafficking muta- 
tions have since been shown to be a common mechanism in 
CHI (Cartier et al, 2001; Taschenberger et al., 2002; Crane and 
Aguilar-Bryan, 2004; Tornovsky et al, 2004; Yan et al, 2004, 2007; 
Marthinet et al, 2005; Muzyamba et al, 2007; Fukuda et al, 201 1; 
Powell et al., 2011; Faletra et al., 2013), in which loss of Katp 
channels at the cell surface results in unregulated insulin secretion 
following constitutive depolarization of the P-cell. 

Among the mutations documented, A116P- and V187D- 
SUR1, both located in TMDO, exhibited reduced association 
with Kir6.2 in co-immunoprecipitation experiments (Chan et al., 
2003), supporting a role of TMDO in subunit-subunit inter- 
actions. Further, AF1388-, A116P-SUR1 and W91R-Kir6.2 all 
showed accelerated degradation (Crane and Aguilar-Bryan, 2004; 
Yan et al., 2004, 2005). These observations are consistent with 
the mutant proteins being misfolded and/or unable to form 
channel complexes. Of note, the aforementioned studies were 
all conducted in mammalian cells rather than Xenopus oocytes 
as oocytes cultured at a lower temperature have less stringent 



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December 2013 | Volume 4 | Article 386 | 7 



Martin et al. 



Correcting Katp channel trafficking defects 



Table 1 | Congenital Hyperinsulinism-associated Katp channel 
trafficking mutations and response to pharmacological rescue 1 . 



Mutation 


Domain 


Rescue 


Rescue 


Gating 


References 






by SU 


by CBZ 


property 








G7R 


TMDO 


Yes 


Yes 


Normal 


Yan etal., 2007 


N24K 


TMDO 


Yes 


Yes 


Normal 


Yan etal., 2007 


F27S 


TMDO 


Yes 


Yes 


Normal 


Yan etal., 2007 


R74W 


TMDO 


Yes 


Yes 


ATP- 


Yan et al., 2007 










insensitive 




A116P 


TMDO 


Yes 


Yes 


Normal 


Yan et al., 2004 


E128K 


TMDO 


Yes 


Yes 


ATP- 


Yan etal., 2007 










insensitive 




V187D 


TMDO 


Yes 


Yes 


Normal 


Yan etal., 2004 


R495Q 


TMD1 


Yes 


Yes 


Unknown 


Yan etal., 2007 


E501K 


TMD1 


Yes 


Yes 


Unknown 


Yan etal., 2007 


L503P 


TMD1 


No 


No 


Unknown 


Yan et al., 2007 


F686S 


NBD1 


No 


No 


Unknown 


Yan etal., 2007 


G716V 


NBD1 


No 


No 


Unknown 


Yan etal., 2007 


E1324K 


TMD2 


N.D. 3 


N.D. 


Normal 


Faletra et al., 












2013 


L1350Q 


NBD2 


No 


No 


Unknown 


Yan etal., 2007 


AF1388 2 


NBD2 


No 


No 


MgADP- 


Cartier et al., 










insensitive 


2001 


M1395R 


NBD2 


N.D. 


N.D. 


Unknown 


Faletra et al., 












2013 


R1419H 


NBD2 


No 


No 


Unknown 


Tornovsky et al., 












2004 


R1437Q 


NBD2 


No 


No 


Unknown 


Muzyamba 












etal., 2007 


D1472H 


NBD2 


No 


No 


Unknown 


Yan etal., 2007 


R1494W 


NBD2 


No 


No 


Unknown 


Tornovsky et al., 












2004 


L1544P 2 


NBD2 


No 


No 


MgADP- 


Taschenberger 










insensitive 


et al., 2002 




W91R 


M1 


N.D. 


N.D. 


Unknown 


Crane and 












Aguilar-Bryan, 












2004 


H259R 


C-term 


N.D. 


N.D. 


Unknown 


Marthinet et al., 












2005 


E282K 


C-term 


N.D. 


N.D. 


Unknown 


Taneja et al., 












2009 


R301G 


C-term 


N.D 


N.D 


Inactivation 4 


Lin etal., 2008 


R301H 


C-term 


N.D 


N.D 


Inactivation 


Lin etal., 2008 


R301P 


C-term 


N.D 


N.D 


Inactivation 


Lin etal., 2008 



1 Only published mutations that have been tested for surface expression are 
included. 

2 These mutations were rescued to the cell surface by mutating the RKR ER 
retention signal to AAA. 

3 N.D.: Not determined. 

4 Inactivation: Spontaneous current decay in the absence of inhibitory ATP 

ER quality control and allow surface expression of mutant chan- 
nels that would otherwise be retained intracellularly (Drumm 
et al., 1991). In addition to protein misfolding, recently a Kir6.2 
mutation E282K identified in a case of histological focal CHI 
(due to combination of a paternal Katp mutation and clonal 



loss of heterozygosity of llpl5) was reported to diminish sur- 
face expression of Katp channels by disrupting a di-acidic ER 
exit signal in Kir6.2 involved in concentration of the channel pro- 
tein into COPII vesicles without affecting channel protein folding 
(Taneja et al, 2009). Also, the SUR1 mutation R1394H report- 
edly causes retention of the channel in the Golgi compartment 
of HEK239 cells, thereby preventing surface expression (Partridge 
et al., 2001). Although increased internalization and degradation 
of surface channels may also reduce the number of channels at the 
cell surface no such examples have been reported. 

CORRECTION OF K AT p CHANNEL TRAFFICKING DEFECTS BY 
PHARMACOLOGICAL CHAPER0NES 

The discovery that protein misfolding is an important cause of 
various diseases has prompted intense investigation into ways of 
overcoming the biogenesis defect as a means of therapy (Powers 
et al., 2009). A key finding came from Denning et al, who showed 
that cells expressing AF508-CFTR had greater surface levels of 
the channel when grown at reduced temperature (Denning et al., 
1992). This demonstrated that manipulation of the cellular fold- 
ing environment can positively impact the biogenesis of proteins. 
Certain exogenous compounds, like glycerol or DMSO, were also 
shown to enhance the expression of trafficking-impaired proteins, 
acting as chemical chaperones (Brown et al., 1996). It is thought 
these compounds interact directly with the protein during folding 
and assembly in the ER and impact biogenesis by one or several of 
the following mechanisms: (1) reducing degradation by thermo- 
dynamically stabilizing a misfolded conformation of the protein; 
(2) increasing the folding rate by stabilizing a folding interme- 
diate; (3) decreasing the misfolding rate by stabilizing the native 
state. Alternatively, chemical chaperones could indirectly affect 
biogenesis of proteins by interacting with endogenous molecular 
chaperones in the ER. 

Chemical chaperones, such as glycerol, are nonspecific, how- 
ever, and enhance the expression of multiple proteins in a cell. 
In a seminal study, Loo et al. demonstrated that pharmacolog- 
ical ligands can specifically promote the stability and expres- 
sion of trafficking-impaired mutant isoforms of P-glycoprotein 
(P-gp), a multidrug resistant protein (Loo and Clarke, 1997). 
Multiple drug compounds known to directly interact with P-gp 
rapidly enhanced expression of various engineered P-gp traf- 
ficking mutants in a dose-dependent manner, producing func- 
tional proteins at the plasma membrane. These results were soon 
echoed by various other groups using high-affinity ligands for 
lysosomal a-galactosidase A (Fan et al., 1999), the V2 vaso- 
pressin receptor (Morello et al, 2000), the HERG (human ether- 
a-go-go-related gene) potassium channel (Zhou et al., 1999), 
among others. An emerging theme began to develop in which 
specific chemical chaperones, termed pharmacological chaper- 
ones, could be administered therapeutically in order to reverse 
trafficking defects found in patients with complex diseases. 
High-throughput screens have identified more advanced com- 
pounds which can rescue mutants with more subtle trafficking 
defects. Several promising hits are currently undergoing clini- 
cal trials for treatment of lysosomal storage diseases, including 
Fabry's, Pombe's, Tay-Sachs, and Gaucher 's diseases, as well as 
CF. Discussed below are recent studies describing the functional 



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December 2013 | Volume 4 | Article 386 | 8 



Martin et al. 



Correcting Katp channel trafficking defects 



restoration of trafficking-impaired Katp channels with the use of 
small molecule correctors. These studies have added to our fun- 
damental understanding of the biogenesis of Katp channels while 
bringing forth a new therapeutic route for treating CHI. Further, 
they have led to new understanding in the ways that pharma- 
cological ligands interact with Katp channels and impact their 
function. 

CORRECTION OF K ATP CHANNEL TRAFFICKING DEFECTS BY 
SULFONYLUREAS 

As with many diseases, a major challenge has been to define 
how mutations in Katp channel subunits associated with CHI 
promote disease. Some mutant channels had been shown to 
be unresponsive to MgADP stimulation (Nichols et al., 1996b; 
Shyng et al, 1998; Snider et al., 2013), while others produce non- 
functional truncated proteins due to insertion of a stop codon 
(Nestorowicz et al., 1997, 1998). An important discovery came 
from Cartier et al., who showed that one Katp channel mutation 
identified in CHI, AF1388 in SUR1, prevented surface expres- 
sion of the channel when expressed in COS cells (Cartier et al., 
2001). Interestingly, functional channel complexes were observed 
when the ER retention signal RKR in AF1388-SUR1 was mutated 
to AAA, which can often allow some trafficking-impaired pro- 
teins to escape the ER. This placed some forms of CHI now in 
the same category as CF and other protein misfolding diseases, 
whereby loss-of-function results from mislocalization of mutant 
proteins. Subsequent work identified additional SUR1 mutations 
in CHI patients that impair the proper trafficking of Katp chan- 
nels, including L1544R A116P, and V187D (Taschenberger et al., 
2002; Yan et al., 2004). 

The successful use of pharmacological ligands in correcting 
trafficking defects of other membrane proteins prompted sev- 
eral groups to apply that same principle to trafficking-impaired 
Katp channels. Yan et al. (2004) demonstrated that two CHI 
mutations, Al 16P and V187D, both located in the first transmem- 
brane domain TMD0 of SUR1, could be rescued by sulfonylureas 
in vitro. Functional study of mutant channels rescued to the cell 
surface by the reversible sulfonylurea tolbutamide revealed nor- 
mal sensitivity to MgADP and ATP once tolbutamide was washed 
out, suggesting that trafficking mutations may not interfere with 
channel function. Presumably, as is the case for pharmacological 
rescue of other trafficking-impaired proteins, sulfonylureas facil- 
itate the biogenesis of these SUR1 mutants by interacting with 
the protein directly during folding and assembly in the ER. Yet an 
understanding of the mechanism of the trafficking impairment 
is key to grasping the mechanism of recovery. Previously, Chan 
et al. showed that TMD0 domain of SUR1 harboring the A116P 
or V187D mutation, had reduced association with Kir6.2 in co- 
immunoprecipitation experiments (Chan et al., 2003). As TMD0 
is known to mediate interactions between SUR1 and Kir6.2, it 
is reasonable to assume that mutations in TMD0 disrupt these 
subunit interactions and prevent channel trafficking out of the 
ER. Yan et al. showed, however, that the trafficking defect in 
A116P and V187D is intrinsic to SUR1. This is based on the 
observation that in the absence of Kir6.2, A116P and V187D 
also prevented Kir6.2-independent surface expression of a SUR1 
protein in which the RKR ER retention signal is inactivated by 



mutation to AAA (SURIaaa)- Another potential explanation for 
the trafficking defect is that these mutations may prevent proper 
shielding of the RKR ER retention signals that must occur dur- 
ing assembly in the ER, as has been demonstrated for the L1544P 
mutation (Taschenberger et al., 2002). Yet mutation of these sig- 
nals in both subunits also failed to improve surface expression of 
the A116P or V187D mutants. These results suggest that SUR1 
misfolding, which also likely adversely affects association with 
Kir6.2, prevents exit of these mutants from the ER. Consistent 
with this notion, channel trafficking defects caused by Al 16P and 
V187D could be overcome by culturing cells at lower temperature 
(Yang et al., 2005), a condition known to facilitate protein folding. 
Metabolic pulse-chase experiments demonstrated that gliben- 
clamide slowed A116P-SUR1 degradation even in the absence 
of Kir6.2 and promoted maturation of the mutant SUR1 when 
Kir6.2 was co-expressed (Yan et al., 2004), providing evidence 
that sulfonylureas facilitate folding and/or prevent misfolding of 
mutant channels during assembly in the ER. 

The question remained, however, of whether sulfonylureas 
act as true pharmacological chaperones by promoting biogene- 
sis through direct binding of the channel. Compelling evidence 
came from a study showing that mutation of residues critical for 
sulfonylurea and glinide binding abolished or reduced these com- 
pounds' ability to rescue Katp channel trafficking mutants (Yan 
et al., 2006). Binding of tolbutamide, a first generation sulfony- 
lurea, has been shown to depend on S1238 in SUR1, representing 
site A (Ashfield et al, 1999). Accordingly, mutation of S1238 to 
tyrosine abolished tolbutamide rescue of SUR1 mutants A116P 
and V187D. Glibenclamide binding involves both site A (S1238) 
and site B, which includes residue Y230 (Bryan et al., 2004). 
Mutation of either site A (S1238Y) or site B (Y230A) dimin- 
ished glibenclamide's rescue effect, while simultaneous mutation 
of both completely abolished it, suggesting that the sulfonylurea 
and benzamido moieties both contribute to the rescue effect of 
glibenclamide. Interestingly, the site B mutation Y230A, in addi- 
tion to attenuating the effect of glibenclamide and abolishing the 
effect of rapaglinide, also rendered tolbutamide ineffective at res- 
cuing mutant channels. This suggests that either Y230 is involved 
in tolbutamide binding or Y230 is necessary for post-binding 
events involved in tolbutamide rescue, such as coupling of SUR1 
and Kir6.2 subunits. In support of the latter, Y230 has been shown 
to be in close proximity to the N-terminus of Kir6.2 (Vila-Carriles 
et al., 2007). Further, deletion of the Kir6.2 N-terminus abol- 
ishes tolbutamide channel block in membrane patches (Koster 
et al., 1999; Reimann et al., 1999), suggesting a functional inter- 
face at Y230 of SUR1 and the N-terminus of Kir6.2 that couples 
tolbutamide binding to changes in channel activity. The results 
of S1238Y and/or Y230A mutants on the ability of sulfonylureas 
to rescue Katp trafficking mutants were also in parallel to their 
ability to block channel activity. Thus, there is likely a common 
mechanism in transducing ligand binding to functional outcome. 
Finally, a key finding from this study is that sulfonylureas act 
on the channel complex, rather than SUR1 alone, to restore sur- 
face expression of trafficking mutants, as pharmacological rescue 
by sulfonylureas is dependent on Kir6.2. Expression of either 
A116Paaa-SUR1 or V187Daaa-SUR1, which lack the RKR ER 
retention motif, could not be rescued without co-expression of 



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December 2013 | Volume 4 | Article 386 | 9 



Martin et al. 



Correcting Katp channel trafficking defects 



Kir6.2. The requirement of Kir6.2 for the rescue effect could be 
explained by the involvement of Kir6.2 in sulfonylurea binding to 
SUR1; alternatively, Kir6.2 could participate in the tertiary folding 
of mutant SUR1 subunits. 

An interesting trend noted for all Katp trafficking mutants 
tested thus far is that only mutations within TMDO of SUR1 are 
amenable to pharmacological rescue by sulfonylureas (Table 1). 
TMDO, the first of three transmembrane domains in SUR1, is a 
unique structural feature not shared by most ABC transporters, 
including CFTR or P-gp, which contain only TMD1 and TMD2. 
When expressed alone, TMDO has been shown to physically asso- 
ciate with Kir6.2 and both facilitate its expression and modulate 
its gating function, making this domain a distinct functional 
entity (Babenko and Bryan, 2003; Chan et al., 2003). The studies 
described above demonstrate that sulfonylureas exert chaperon- 
ing effects by binding regions downstream of TMDO. Thus, ligand 
binding to the core ABC structure is likely translated into struc- 
tural and functional outcomes at TMDO to overcome trafficking 
defects caused by mutations within this region. Such substrate- 
induced transmembrane domain interactions have been reported 
previously: in human P-gp, also an ABC transporter, substrate 
binding promoted superfolding of partially folded intermediates 
via interactions between the two transmembrane domains TMD1 
and TMD2 (Loo and Clarke, 1998). As such, a likely mecha- 
nism for the pharmacological chaperone effect is that sulfonylurea 
binding to the SUR1/Kir6.2 complex induces structural changes 
in TMDO that restore functional interactions between SUR1 and 
Kir6.2, allowing trafficking of the channel out of the ER. The pre- 
cise nature of the interface between TMDO and Kir6.2 is unclear, 
however. Interestingly, a recent study by Zhou et al. showed that 
a point mutation in Kir6.2, Q52E, located in the N-terminus of 
the protein just before the slide helix, partially compensated for 
the trafficking defects caused by SUR1-TMD0 mutations F27S 
and A116P, indicating that altered molecular interactions with 
Kir6.2 can overcome impaired channel folding/assembly caused 
by TMDO mutations (Zhou et al., 2013). An understanding of 
how these domains are interacting will provide valuable insight 
into not only the mechanism of pharmacological rescue, but also 
how physiological or pharmacological interactions at SUR1 are 
coupled to changes in channel activity at Kir6.2. 

CORRECTION OF K ATP CHANNEL TRAFFICKING DEFECTS BY 
AF508-CFTR CORRECTORS, IN PARTICULAR BY CARBAMAZEPINE 

The fact that multiple TMDO trafficking mutants have nor- 
mal responses to metabolic signals and pharmacological ligands 
provides proof of principle that pharmacological rescue is a ther- 
apeutically viable alternative to the current treatment for many 
CHI with such mutations, which often relies on partial or near 
complete removal of the pancreas. Translation of these findings, 
however, has been hindered by the pharmacology of gliben- 
clamide, the most effective sulfonylurea at rescuing trafficking- 
impaired Katp channels. SUR1 binds glibenclamide with high 
affinity and slow dissociation kinetics, resulting in an irreversible 
block on channel function; rescued channels would therefore be 
unable to hyperpolarize the fl-cell in order to attenuate insulin 
release. Tolbutamide, another sulfonylurea that effectively res- 
cues multiple TMDO trafficking mutants, binds Katp channels 



reversibly. A study released by the University Group Diabetes 
Program, however, implicated tolbutamide in increased mortality 
secondary to cardiovascular events (Schwartz and Meinert, 2004). 
Thus, in terms of therapy, there is a need for additional com- 
pounds that promote robust recovery of Katp channel trafficking 
mutants and bind reversibly, but are also safe for administration 
to patients. 

Like trafficking-impaired Katp channels in CHI, AF508-CFTR 
causes partial misfolding of the channel and clearance through 
the ubiquitin/proteasome pathway, resulting in CF. Much effort 
has been devoted to identifying small molecules that correct 
this trafficking defect, and high-throughput drug screens have 
yielded several promising compounds (Pedemonte et al., 2005; 
Carlile et al, 2007). As CFTR and SUR1 are both members 
of the ABC transporter superfamily and have common struc- 
tural features in the ABC core domain, it is reasonable to 
hypothesize that small molecules which correct folding and traf- 
ficking defects in AF508-CFTR might also rescue trafficking- 
impaired Katp channels caused by mutations in SUR1. Powell 
et al. first reported the effects of compounds known to stimu- 
late CFTR trafficking on human fi-cells lacking functional Katp 
currents from CHI patients harboring various ABCC8 muta- 
tions (Powell et al., 2011). Although it remains unknown how 
these mutations impact channel trafficking and gating to cause 
loss of channel activity, the study demonstrated that a AF508- 
CFTR corrector, 4-phenylbutyrate, could recover activities of 
Katp channels in fi-cells isolated from a patient bearing the 
SUR1 compound heterozygous mutation Arg998X/Serl449dup 
(Powell et al., 2011). Additionally, the study showed that incu- 
bation of P-cells from another patient bearing homozygous 
ABCC8 intronic mutation c.1467+5G>A with a combination 
of 3-isobutyl-l-methylxanthine (IBMX), forskolin, and phorbol 
myristic acid (PMA), compounds expected to activate PKA and 
PKC, for 1 h also led to increased channel activity. Because PKA 
activation has been reported to promote trafficking of several 
ion channels to the cell surface whereas PKC activation has been 
reported to reduce surface Katp expression by diverting endocy- 
tosed channels to lysosomal degradation (Manna et al., 2010), 
the authors speculate that the positive effect of PKA activation 
likely overrides the negative effects of PKC to lead to an over- 
all increase in surface expression of Katp channels (Powell et al., 
2011). It is interesting to note that a role of PKA in Katp chan- 
nel trafficking has indeed been reported. A study by Yang et al. 
showed that glucose stimulation recruits fi-cell Katp channels to 
the cell surface in a PKA-dependent manner (Yang et al, 2007). 
In addition, Chen et al. showed that PKA activation promotes 
Katp channel trafficking to the surface in INS-1 rat insulinoma 
cells by promoting F-actin depolymerization without affecting 
overall channel protein levels (Chen et al, 2013a). The find- 
ings by Powell et al. will undoubtedly stimulate future research 
to harness the therapeutic potential of these compounds and to 
understand the molecular mechanisms by which these molecules 
restore functional expression of Katp channels in CHI patients. 

A more recent study by Sampson et al. tested the effects of 
multiple CFTR correctors identified in a chemical library screen 
(Carlile et al, 2007) on the processing efficiency of two SUR1 
trafficking mutants, A116P and V187D (Sampson et al, 2013). 



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Martin et al. 



Correcting Katp channel trafficking defects 



Among the compounds that improved the processing efficiency of 
the two mutant SUR1 proteins is the drug carbamazepine (CBZ). 
CBZ has been used for decades in the treatment of epilepsy, neu- 
ropathic pain, as well as mental illnesses like bipolar disorder. 
CBZ has a well-documented role as a sodium channel blocker, 
but studies have shown effects on calcium channels as well as 
GABA receptors (Ambrosio et al., 2002). Our group focused 
attention on CBZ as a potential pharmacological chaperone for 
SUR1 trafficking mutants, as it is approved by the Food and 
Drug Administration and has an established biosafety profile. 
In a recent report we demonstrated that CBZ effectively res- 
cues multiple Katp trafficking mutations previously identified 
in CHI (Chen et al., 2013b) (Figure 3C). Interestingly, as with 
sulfonylureas, only mutations present in TMDO of SUR1 were 
amenable to pharmacological rescue by CBZ. This suggests sul- 
fonylureas and CBZ may act through a common mechanism 
to enhance surface expression of trafficking mutants, such as 
by stabilizing interactions between TMDO and Kir6.2. There are 
studies showing, however, that CBZ can induce autophagy to 
facilitate clearance of misfolded protein aggregates (Sarkar et al., 
2007; Hidvegi et al, 2010), which could in turn alleviate ER 
stress and promote surface expression of trafficking mutants. This 
raises the possibility that CBZ may enhance surface expression 
of TMDO trafficking mutants not by acting as a pharmacologi- 
cal chaperone but by inducing autophagy. Disfavoring this idea, 
inhibition of autophagy by chloroquine or Ly294002 did not 
block CBZ's ability to rescue Katp trafficking mutants. Moreover, 
stimulation of autophagy by rapamycin or Li + did not enhance 
mutant Katp channel processing or expression. These results indi- 
cate that CBZ most likely rescues mutant channels through an 
autophagy-independent pathway. 

A surprising and intriguing finding by Chen et al. is that CBZ 
inhibits the activity of mutant channels rescued to the cell sur- 
face, as demonstrated in 86 Rb + efflux experiments. Subsequent 
electrophysiology experiments showed that CBZ inhibits chan- 
nel activity when applied to the cytoplasmic face of isolated 
membrane patches containing wild-type Katp channels. These 
observations suggest that CBZ may act as a Katp channel antago- 
nist, like sulfonylureas. Further characterization of the inhibitory 
effect of CBZ by 86 Rb + efflux assays showed that the function 
of rescued mutant channels could be completely recovered after 
extensive washout (~90min), similar to that observed for the 
reversible, low-affinity sulfonylurea tolbutamide but not the irre- 
versible high affinity sulfonylurea glibenclamide (Yan et al, 2004). 
Because the effect of CBZ on mutant channel trafficking could be 
detected at a concentration as low as 0.2(i,M (Chen et al., 2013b) 
(see Figure 3C), it is conceivable that low doses of CBZ could be 
used to rescue mutant channels to the cell surface without potent 
inhibition of channel activity. Another approach to circumvent 
the problem of chaperone inhibitors may be to simultaneously 
apply correctors and compounds that boost the function of res- 
cued proteins, referred to as potentiators (Rowe and Verkman, 
2013). Indeed, we have found that while diazoxide is unable to 
activate channels rescued by the irreversible antagonist gliben- 
clamide, it significantly increased the activity of channels rescued 
by CBZ in Rb efflux assays. In these experiments, CBZ was not 
included in the 40-min efflux period so some CBZ was likely to 



have dissociated from the rescued channels, as evidenced by a 
small increase in efflux even without diazoxide; however, inclu- 
sion of diazoxide during efflux further increased channel activity, 
indicating that diazoxide can facilitate functional recovery of CBZ 
rescued channels. Clinically, this has important implications, as it 
has been proposed that small changes in Katp channel activity are 
correlated with large differences in clinical outcome (Macmullen 
et al., 2011). Interestingly, we have found that diazoxide pre- 
cludes glibenclamide's ability to rescue Katp trafficking mutants, 
while it has no effect on the rescue of mutant channels by CBZ 
when the drugs are co-administered. This further supports the 
clinical feasibility of a combination therapy using a pharmaco- 
logical chaperone and a channel activator to alleviate symptoms 
in patients with CHI. Also important, the effects of CBZ were 
observed in two physiologically relevant systems, namely the rat 
P-cell line INS-1 and freshly isolated primary human P-cells. 
Further, the concentrations at which CBZ is effective at rescuing 
TMDO trafficking mutants (10-50 |iM) is similar to those used to 
block Na + channels, suggesting the approved dosage prescribed 
for CBZ will impact Katp channel expression. These data make 
a compelling case for further exploration of CBZ as a potential 
treatment for patients with certain forms of CHI. 

Besides therapeutic implications, CBZ and glibenclamide dif- 
fer markedly in their chemical structures and yet both rescue the 
expression of only those Katp channels with trafficking mutations 
in TMDO of SUR1 and both inhibit K A tp channel activity. An 
intriguing question to address in the future is whether the closed 
channel conformation rendered by these ligands represents a state 
that favors forward trafficking. 

THE ROLE OF DIAZOXIDE ON K AT p CHANNEL TRAFFICKING 

Ideally, a pharmacological chaperone would correct protein traf- 
ficking defects without compromising or even enhance protein 
function. In this regard, it is worth noting that the Katp channel 
opener diazoxide has been reported to correct channel traffick- 
ing defects caused by the SUR1 mutations R1394H (Partridge 
et al., 2001). Using a stable HEK293 cell line co-expressing wild- 
type or R1394H His-tagged hamster SUR1 and Kir6.2 tagged 
at the C-terminus with a HMA (heart muscle kinase phospho- 
rylation site) -FLAG epitope, Partridge et al. showed that the 
mutant SUR1 failed to reach the cell surface and instead accu- 
mulated in the trans-Golgi network, and that diazoxide was 
able to restore surface expression of the R1394H mutant SUR1. 
However, a subsequent study by Yan et al. using FLAG-tagged 
R1394H mutant hamster SUR1 transiently co-expressed with 
Kir6.2 in COS cells reported normal trafficking of the mutant 
to the cell surface (Yan et al., 2004). Whether these different 
results are due to the different constructs or cells used remain 
to be resolved. Aside from the R1394H mutation reported by 
Partridge et al., no other known trafficking mutations tested so 
far are rescued by diazoxide (Yan et al, 2004, 2007; Chen et al., 
2013b). Also worth noting, Powell et al. showed that a diazox- 
ide analog BPDZ 154 restored ATP- inhibited channel activity in 
human fi-cells from a CHI patient with the homozygous ABCC8 
intronic mutation c.1467+5G>A after 24-48 h incubation. Since 
it is not clear how this intronic mutation affects channel traf- 
ficking and/or function, it remains to be determined whether 



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Martin et al. 



Correcting Katp channel trafficking defects 



BPDZ 154 enhances channel trafficking and/or gating to restore 
function. 

INTERPLAY BETWEEN CHANNEL EXPRESSION AND GATING 
IN DISEASE MANIFESTATION 

Although some TMDO mutations only impair channel trafficking 
such that pharmacological rescue of mutant channels to the cell 
surface is expected to partially or fully restore channel function, 
some impact both channel biogenesis and gating as exemplified 
by the R74W and E128K mutations (Pratt et al, 2009). These 
mutations result in CHI by preventing channel expression at 
the cell surface. Upon rescue to the cell surface by tolbutamide 
followed by washout, mutant channels exhibit reduced sensitiv- 
ity to ATP inhibition, a gating defect commonly observed in 
gain-of-function mutations associated with permanent neona- 
tal diabetes (PNDM). Detailed analysis revealed that these two 
mutations cause functional uncoupling between SUR1 and Kir6.2 
by impairing the ability of SUR1 to hypersensitize Kir6.2 chan- 
nels to ATP inhibition (Pratt et al., 2011). When these mutations 
were introduced into the insulinoma cells INS-1 and rescued to 
the cell surface by tolbutamide, subsequent washout of tolbu- 
tamide led to cells with more hyperpolarized membrane potential 
in the face of glucose stimulation (Pratt et al., 2009), similar 
to P-cells with gain-of-function, diabetes-causing mutant Katp 
channels. 

Intuitively, trafficking defects are associated with loss of chan- 
nel function and the disease CHI. However, it has been shown that 
many mutations identified in PNDM (Gloyn et al., 2004; Proks 
et al, 2004, 2005, 2006; Koster et al, 2005) also reduce chan- 
nel biogenesis efficiency, including Q52R, V59G/M, R201C/H 
and I296L in Kir6.2 (Lin et al, 2006a) as well as F132L in 
SUR1 (Pratt et al., 2009) when expressed heterologously as 
homomeric mutant channels (Table 2). It is interesting that 
glibenclamide was also found to significantly improve surface 
expression of heterologously expressed homomeric mutant chan- 
nels (Lin et al., 2006a), again hinting at SUR1-Kir6.2 interactions 
in sulfonylurea-mediated rescue. Whether CBZ also improves 
surface expression of these PNDM mutations remains to be 
determined. Because these PNDM mutations are dominant, het- 
erozygous mutations with severe gating defects, a mutant channel 
subunit can exert its gain-of-function gating effect by co-assembly 
with the WT allele. In this scenario, the extent of expression 
of mutant subunit in the cell surface channel population may 
determine the extent of overall channel gating defect and thus, 
disease severity as has been proposed for several PNDM muta- 
tions, including V324M in SUR1 (Zhou et al, 2010) as well as 
C42R and Pro226-Pro232 deletion mutation in Kir6.2 (Yorifuji 
et al, 2005; Lin et al., 2013). 

The above studies highlight the importance of the interplay 
between channel expression and gating defects in determining 
disease phenotype. As Katp conductance is a product of the num- 
ber of channels in the fi-cell membrane and the open probability 
of the channel at a given metabolic state dictated by channel gat- 
ing properties, correlation between channel defects and disease 
phenotype would require thorough analysis of the impact of a 
mutation on both channel expression and channel gating as well 
as consideration of the genetic context of the mutation. 



Table 2 | Neonatal Diabetes-associated Katp channel trafficking 
mutations and response to sulfonylurea treatment. 



Mutation Surface expression Gating References 
increased by SU property 



SUR1 


F132L 


Yes 


Increased P G 


Pratt et al., 2009 


V324M 


N.D. 


Increased MgADP 


Zhou et al., 2010 






sensitivity 






C42R 


N.D. 


Increased P 0 


Yorifuji et al., 2005 


Q52R 


Yes 


Increased P 0 


Proks et al., 2004; 








Lin et al., 2006a 


V59G 


Yes 


Increased P 0 


Proks et al., 2004; 








Lin et al., 2006a 


V59M 


Yes 


Increased P G 


Koster et al., 2005; 








Lin et al., 2006a 


R201C 


Yes 


Decreased ATP 


Proks et al., 2004; 






inhibition 


Lin et al., 2006a 


R201H 


Yes 


Decreased ATP 


Proks et al., 2004; 






inhibition 


Lin et al., 2006a 


Pro226_ 


N.D. 


Increased P D 


Lin et al., 2013 


Pro232del 








I296L 


Yes 


Increased P 0 


Proks et al., 2005; 



Lin et al., 2006a 



CONCLUSIONS AND PERSPECTIVES 

Pharmacological chaperones have emerged as promising thera- 
peutic tools for treating diseases resulting from defective protein 
folding and/or trafficking. Demonstration that sulfonylureas and 
CBZ are effective pharmacological agents able to restore surface 
expression of Katp trafficking mutants identified in congenital 
hyperinsulinism has direct clinical relevance. As CBZ is an FDA- 
approved drug, it may stand to rapidly improve current therapies 
for patients harboring trafficking mutations within TMDO of 
SUR1. In order to spur translation of these findings into real 
treatment, an important next step is to demonstrate the efficacy 
of CBZ in P-cells isolated from CHI patients with Katp traf- 
ficking mutations within TMDO. Also, currently only a subset 
of identified TMDO trafficking mutations associated with dis- 
ease has been examined for their ability to be rescued by CBZ. 
The therapeutic applicability of CBZ for treating Katp traffick- 
ing disorders will likely expand in the future as more mutations 
are identified and tested. Finally, although CBZ also inhibits Katp 
channel function, this inhibition is reversible and can be partially 
overcome by co-application of potentiators, such as diazoxide, 
without compromising CBZ's corrector effect. These findings 
represent significant improvements over pharmacological rescue 
using glibenclamide, but CBZ itself may only be a model demon- 
strating the potential that future pharmacological correctors hold 
for treating Katp channel trafficking disorders. 

Beyond the therapeutic implications, in vitro studies utilizing 
pharmacological chaperones and naturally occurring mutations 
in Katp channel subunits have enhanced our understanding 
of structure-function relationships in terms of biogenesis and 
molecular assembly, as well as gating and coupling between 
subunits. For instance, the finding that only trafficking mutations 



Frontiers in Physiology | Membrane Physiology and Membrane Biophysics 



December 2013 | Volume 4 | Article 386 | 12 



Martin et al. 



Correcting Katp channel trafficking defects 



within TMDO of SUR1 are amenable to pharmacological res- 
cue further underscores the importance of this unique domain 
in mediating subunit interactions with Kir6.2 and highlights a 
role of TMDO in channel assembly. The fact that drug bind- 
ing, at least in the case of sulfonylureas, to LO and TMD2 of 
SUR1 as well as N-terminus of Kir6.2 has functional consequences 
for mutations within TMDO suggests that either these domains 
can physically interact or there is a mechanism for transducing 
structural changes in trans to TMDO. Further, the fact that some 
Katp trafficking mutants rescued by reversible inhibitors (CBZ, 
tolbutamide) are functional upon drug washout and retain nor- 
mal responses to metabolic signals and pharmacological ligands 
implies that these residues in TMDO are not involved in gating or 
other functional aspects of the channel, but likely play important 
roles in the folding of this domain or may even be key residues at 
the interface of SUR1 and Kir6.2. At present, it is unclear whether 
CBZ binds directly to the channel complex during biogenesis or 
impacts channel expression and gating indirectly through interac- 
tions with other proteins. If CBZ does interact directly, defining 
the binding sites on Katp will provide valuable information on 
the mechanism by which this drug modulates channel folding, 
assembly and gating. This knowledge is critically important for 
future efforts to design more effective drugs that will target 
the biogenesis or gating defects of disease-causing mutant Katp 
channels. 

ACKNOWLEDGMENTS 

This work was supported by National Institutes of Health grant 
DK57699 and DK66485 (to Show-Ling Shyng). Gregory Martin 
is supported by an NIH Ruth L. Kirschstein T32 PMCB Training 
Grant. 

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Conflict of Interest Statement: The authors declare that the research was con- 
ducted in the absence of any commercial or financial relationships that could be 
construed as a potential conflict of interest. 

Received: 01 October 2013; accepted: 09 December 2013; published online: 24 
December 2013. 

Citation: Martin GM, Chen P-C, Devaraneni P and Shyng S-L (2013) 
Pharmacological rescue of trafficking-impaired ATP-sensitive potassium channels. 
Front. Physiol. 4:386. doi: 10.3389/fphys.2013.00386 

This article was submitted to Membrane Physiology and Membrane Biophysics, a 
section of the journal Frontiers in Physiology. 

Copyright © 2013 Martin, Chen, Devaraneni and Shyng. This is an open-access 
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