A Quartet of Leucine Residues in the Guanylate Kinase Domain of CaVβ Determines the Plasma Membrane Density of the CaV2.3 Channel

Ca_Vβ subunits are formed by a Src homology 3 domain and a guanylate kinase-like (GK) domain connected through a variable HOOK domain. Complete deletion of the Src homology 3 domain (75 residues) as well as deletion of the HOOK domain (47 residues) did not alter plasma membrane density of Ca_V2.3 nor its typical activation gating. In contrast, six-residue deletions in the GK domain disrupted cell surface trafficking and functional expression of Ca_V2.3. Mutations of residues known to carry nanomolar affinity binding in the GK domain of Ca_Vβ (P175A, P179A, M195A, M196A, K198A, S295A, R302G, R307A, E339G, N340G, and A345G) did not significantly alter cell surface targeting or gating modulation of Ca_V2.3. Nonetheless, mutations of a quartet of leucine residues (either single or multiple mutants) in the α3, α6, β10, and α9 regions of the GK domain were found to significantly impair cell surface density of Ca_V2.3 channels. Furthermore, the normalized protein density of Ca_V2.3 was nearly abolished with the quadruple Ca_Vβ3 Leu mutant L200G/L303G/L337G/L342G. Altogether, our observations suggest that the four leucine residues in Ca_Vβ3 form a hydrophobic pocket surrounding key residues in the α-interacting domain of Ca_V2.3. This interaction appears to play an essential role in conferring Ca_Vβ-induced modulation of the protein density of Ca_Vα1 subunits in Ca_V2 channels.     

Single-channel properties of human NaV1.1 and mechanism of channel dysfunction in SCN1A-associated epilepsy

Mutations in genes encoding neuronal voltage-gated sodium channel subunits have been linked to inherited forms of epilepsy. The majority of mutations (>100) associated with generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI) occur in SCN1A encoding the NaV1.1 neuronal sodium channel alpha-subunit. Previous studies demonstrated functional heterogeneity among mutant SCN1A channels, revealing a complex relationship between clinical and biophysical phenotypes. To further understand the mechanisms responsible for mutant SCN1A behavior, we performed a comprehensive analysis of the single-channel properties of heterologously expressed recombinant WT-SCN1A channels. Based on these data, we then determined the mechanisms for dysfunction of two GEFS+-associated mutations (R1648H, R1657C) both affecting the S4 segment of domain 4. WT-SCN1A has a slope conductance (17 pS) similar to channels found in native mammalian neurons. The mean open time is approximately 0.3 ms in the -30 to -10 mV range. The R1648H mutant, previously shown to display persistent sodium current in whole-cell recordings, exhibited similar slope conductance but had an increased probability of late reopening and a subfraction of channels with prolonged open times. We did not observe bursting behavior and found no evidence for a gating mode shift to explain the increased persistent current caused by R1648H. Cells expressing R1657C exhibited conductance, open probability, mean open time, and latency to first opening similar to WT channels but reduced whole-cell current density, suggesting decreased number of functional channels at the plasma membrane. In summary, our findings define single-channel properties for WT-SCN1A, detail the functional phenotypes for two human epilepsy-associated sodium channel mutants, and clarify the mechanism for increased persistent sodium current induced by the R1648H allele.     

Of cilia,titin, and neurosteroids

Missense mutations at arginine residues in the S4 voltage-sensor domains of Na_V1.4 are an established cause of hypokalemic periodic paralysis, an inherited disorder of skeletal muscle involving recurrent episodes of weakness in conjunction with low serum K⁺. Expression studies in oocytes have revealed anomalous, hyperpolarization-activated gating pore currents in mutant channels. This aberrant gating pore conductance creates a small inward current at the resting potential that is thought to contribute to susceptibility to depolarization in low K⁺ during attacks of weakness. A critical component of this hypothesis is the magnitude of the gating pore conductance relative to other conductances that are active at the resting potential in mammalian muscle: large enough to favor episodes of paradoxical depolarization in low K⁺, yet not so large as to permanently depolarize the fiber. To improve the estimate of the specific conductance for the gating pore in affected muscle, we sequentially measured Na⁺ current through the channel pore, gating pore current, and gating charge displacement in oocytes expressing R669H, R672G, or wild-type Na_V1.4 channels. The relative conductance of the gating pore to that of the pore domain pathway for Na⁺ was 0.03%, which implies a specific conductance in muscle from heterozygous patients of ∼10 µS/cm² or 1% of the total resting conductance.Unexpectedly, our data also revealed a substantial decoupling between gating charge displacement and peak Na⁺ current for both R669H and R672G mutant channels. This decoupling predicts a reduced Na⁺ current density in affected muscle, consistent with the observations that the maximal dV/dt and peak amplitude of the action potential are reduced in fibers from patients with R672G and in a knock-in mouse model of R669H. The defective coupling between gating charge displacement and channel activation identifies a previously unappreciated mechanism that contributes to the reduced excitability of affected fibers seen with these mutations and possibly with other R/X mutations of S4 of Na_V, Ca_V, and K_V channels associated with human disease.     

Structural Pharmacology of Voltage-Gated Sodium Channels

Voltage-gated sodium (Na_V) channels initiate and propagate action potentials in excitable tissues to mediate key physiological processes including heart contraction and nervous system function. Accordingly, Na_V channels are major targets for drugs, toxins and disease-causing mutations. Recent breakthroughs in cryo-electron microscopy have led to the visualization of human Na_V1.1, Na_V1.2, Na_V1.4, Na_V1.5 and Na_V1.7 channel subtypes at high-resolution. These landmark studies have greatly advanced our structural understanding of channel architecture, ion selectivity, voltage-sensing, electromechanical coupling, fast inactivation, and the molecular basis underlying Na_V channelopathies. Na_V channel structures have also been increasingly determined in complex with toxin and small molecule modulators that target either the pore module or voltage sensor domains. These structural studies have provided new insights into the mechanisms of pharmacological action and opportunities for subtype-selective Na_V channel drug design. This review will highlight the structural pharmacology of human Na_V channels as well as the potential use of engineered and chimeric channels in future drug discovery efforts.     

Proton Sensors in the Pore Domain of the Cardiac Voltage-gated Sodium Channel

Protons impart isoform-specific modulation of inactivation in neuronal, skeletal muscle, and cardiac voltage-gated sodium (Na_V) channels. Although the structural basis of proton block in Na_V channels has been well described, the amino acid residues responsible for the changes in Na_V kinetics during extracellular acidosis are as yet unknown. We expressed wild-type (WT) and two pore mutant constructs (H880Q and C373F) of the human cardiac Na_V channel, Na_V1.5, in Xenopus oocytes. C373F and H880Q both attenuated proton block, abolished proton modulation of use-dependent inactivation, and altered pH modulation of the steady-state and kinetic parameters of slow inactivation. Additionally, C373F significantly reduced the maximum probability of use-dependent inactivation and slow inactivation, relative to WT. H880Q also significantly reduced the maximum probability of slow inactivation and shifted the voltage dependence of activation and fast inactivation to more positive potentials, relative to WT. These data suggest that Cys-373 and His-880 in Na_V1.5 are proton sensors for use-dependent and slow inactivation and have implications in isoform-specific modulation of Na_V channels.     

RNA Editing in the Central Cavity as a Mechanism to Regulate Surface Expression of the Voltage-gated Potassium Channel Kv1.1

Voltage-gated potassium (Kv) 1.1 channels undergo a specific enzymatic RNA deamination, generating a channel with a single amino acid exchange located in the inner pore cavity (Kv1.1^I400V). We studied I400V-edited Kv1.1 channels in more detail and found that Kv1.1^I400V gave rise to much smaller whole-cell currents than Kv1.1. To elucidate the mechanism behind this current reduction, we conducted electrophysiological recordings on single-channel level and did not find any differences. Next we examined channel surface expression in Xenopus oocytes and HeLa cells using a chemiluminescence assay and found the edited channels to be less readily expressed at the surface membrane. This reduction in surface expression was verified by fluorescence imaging experiments. Western blot analysis for comparison of protein abundances and glycosylation patterns did not show any difference between Kv1.1 and Kv1.1^I400V, further indicating that changed trafficking of Kv1.1^I400V is causing the current reduction. Block of endocytosis by dynasore or AP180C did not abolish the differences in current amplitudes between Kv1.1 and Kv1.1^I400V, suggesting that backward trafficking is not affected. Therefore, our data suggest that I400V RNA editing of Kv1.1 leads to a reduced current size by a decreased forward trafficking of the channel to the surface membrane. This effect is specific for Kv1.1 because coexpression of Kv1.4 channel subunits with Kv1.1^I400V abolishes these trafficking effects. Taken together, we identified RNA editing as a novel mechanism to regulate homomeric Kv1.1 channel trafficking. Fine-tuning of Kv1.1 surface expression by RNA editing might contribute to the complexity of neuronal Kv channel regulation.     

The β1-Subunit of Nav1.5 Cardiac Sodium Channel Is Required for a Dominant Negative Effect through α-α Interaction

Brugada syndrome (BrS) is an inherited autosomal dominant cardiac channelopathy. Several mutations on the cardiac sodium channel Na_v1.5 which are responsible for BrS lead to misfolded proteins that do not traffic properly to the plasma membrane. In order to mimic patient heterozygosity, a trafficking defective mutant, R1432G was co-expressed with Wild Type (WT) Na_v1.5 channels in HEK293T cells. This mutant significantly decreased the membrane Na current density when it was co-transfected with the WT channel. This dominant negative effect did not result in altered biophysical properties of Na_v1.5 channels. Luminometric experiments revealed that the expression of mutant proteins induced a significant reduction in membrane expression of WT channels. Interestingly, we have found that the auxiliary Na channel β₁-subunit was essential for this dominant negative effect. Indeed, the absence of the β₁-subunit prevented the decrease in WT sodium current density and surface proteins associated with the dominant negative effect. Co-immunoprecipitation experiments demonstrated a physical interaction between Na channel α-subunits. This interaction occurred only when the β₁-subunit was present. Our findings reveal a new role for β₁-subunits in cardiac voltage-gated sodium channels by promoting α-α subunit interaction which can lead to a dominant negative effect when one of the α-subunits shows a trafficking defective mutation.     

Membrane Trafficking of Large Conductance Calcium-activated Potassium Channels Is Regulated by Alternative Splicing of a Transplantable, Acidic Trafficking Motif in the RCK1-RCK2 Linker

Trafficking of the pore-forming α-subunits of large conductance calcium- and voltage-activated potassium (BK) channels to the cell surface represents an important regulatory step in controlling BK channel function. Here, we identify multiple trafficking signals within the intracellular RCK1-RCK2 linker of the cytosolic C terminus of the channel that are required for efficient cell surface expression of the channel. In particular, an acidic cluster-like motif was essential for channel exit from the endoplasmic reticulum and subsequent cell surface expression. This motif could be transplanted onto a heterologous nonchannel protein to enhance cell surface expression by accelerating endoplasmic reticulum export. Importantly, we identified a human alternatively spliced BK channel variant, hSloΔ_579–664, in which these trafficking signals are excluded because of in-frame exon skipping. The hSloΔ_579–664 variant is expressed in multiple human tissues and cannot form functional channels at the cell surface even though it retains the putative RCK domains and downstream trafficking signals. Functionally, the hSloΔ_579–664 variant acts as a dominant negative subunit to suppress cell surface expression of BK channels. Thus alternative splicing of the intracellular RCK1-RCK2 linker plays a critical role in determining cell surface expression of BK channels by controlling the inclusion/exclusion of multiple trafficking motifs.     

C Terminus of Nucleotide Binding Domain 1 Contains Critical Features for Cystic Fibrosis Transmembrane Conductance Regulator Trafficking and Activation

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl⁻ channel physiologically important in fluid-transporting epithelia and pathologically relevant in several human diseases. Here, we show that mutations in the C terminus of the first nucleotide binding domain comprising the latest β strands (β_c5 and β_c6) influence the trafficking, channel activity, and pharmacology of CFTR. We mutated CFTR amino acids located in the β_c5-β_c6 hairpin, within the β_c5 strand (H620Q), within the β-turn linking the two β strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the β_c6 strand. Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. For G622D and G628R, the abnormal activity is likely due to a defective maturation process, as assessed by the augmented activity and mature C-band observed in the presence of the trafficking corrector miglustat. In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Finally, G622D and G628R were activated by the CFTR agonists genistein, RP-107, and isobutylmethylxanthine. Our results identify the C terminus of the CFTR first nucleotide binding domain as an important molecular site for the trafficking of CFTR protein, for the control of CFTR channel gating, and for the pharmacological effect of a dual activity agent.     

A Heterozygous Deletion Mutation in the Cardiac Sodium Channel Gene SCN5A with Loss- and Gain-of-Function Characteristics Manifests as Isolated Conduction Disease,without Signs of Brugada or Long QT Syndrome

Background

The SCN5A gene encodes for the α-subunit of the cardiac sodium channel Na_V1.5, which is responsible for the rapid upstroke of the cardiac action potential. Mutations in this gene may lead to multiple life-threatening disorders of cardiac rhythm or are linked to structural cardiac defects. Here, we characterized a large family with a mutation in SCN5A presenting with an atrioventricular conduction disease and absence of Brugada syndrome.

Method and Results

In a large family with a high incidence of sudden cardiac deaths, a heterozygous SCN5A mutation (p.1493delK) with an autosomal dominant inheritance has been identified. Mutation carriers were devoid of any cardiac structural changes. Typical ECG findings were an increased P-wave duration, an AV-block I° and a prolonged QRS duration with an intraventricular conduction delay and no signs for Brugada syndrome. HEK293 cells transfected with 1493delK showed strongly (5-fold) reduced Na⁺ currents with altered inactivation kinetics compared to wild-type channels. Immunocytochemical staining demonstrated strongly decreased expression of SCN5A 1493delK in the sarcolemma consistent with an intracellular trafficking defect and thereby a loss-of-function. In addition, SCN5A 1493delK channels that reached cell membrane showed gain-of-function aspects (slowing of the fast inactivation, reduction in the relative fraction of channels that fast inactivate, hastening of the recovery from inactivation).

Conclusion

In a large family, congregation of a heterozygous SCN5A gene mutation (p.1493delK) predisposes for conduction slowing without evidence for Brugada syndrome due to a predominantly trafficking defect that reduces Na⁺ current and depolarization force.     

Gating pore currents occur in CaV1.1 domain III mutants associated with HypoPP

Mutations in the voltage sensor domain (VSD) of Ca_V1.1, the α_1S subunit of the L-type calcium channel in skeletal muscle, are an established cause of hypokalemic periodic paralysis (HypoPP). Of the 10 reported mutations, 9 are missense substitutions of outer arginine residues (R1 or R2) in the S4 transmembrane segments of the homologous domain II, III (DIII), or IV. The prevailing view is that R/X mutations create an anomalous ion conduction pathway in the VSD, and this so-called gating pore current is the basis for paradoxical depolarization of the resting potential and weakness in low potassium for HypoPP fibers. Gating pore currents have been observed for four of the five Ca_V1.1 HypoPP mutant channels studied to date, the one exception being the charge-conserving R897K in R1 of DIII. We tested whether gating pore currents are detectable for the other three HypoPP Ca_V1.1 mutations in DIII. For the less conserved R1 mutation, R897S, gating pore currents with exceptionally large amplitude were observed, correlating with the severe clinical phenotype of these patients. At the R2 residue, gating pore currents were detected for R900G but not R900S. These findings show that gating pore currents may occur with missense mutations at R1 or R2 in S4 of DIII and that the magnitude of this anomalous inward current is mutation specific.     

Electrophysiological Characteristics of a SCN5A Voltage Sensors Mutation R1629Q Associated With Brugada Syndrome

Brugada syndrome (BrS) is an inherited arrhythmogenic syndrome leading to sudden cardiac death, partially associated with autosomal dominant mutations in SCN5A, which encodes the cardiac sodium channel alpha-subunit (Na_v1.5). To date some SCN5A mutations related with BrS have been identified in voltage sensor of Na_v1.5. Here, we describe a dominant missense mutation (R1629Q) localized in the fourth segment of domain IV region (DIV-S4) in a Chinese Han family. The mutation was identified by direct sequencing of SCN5A from the proband’s DNA. Co-expression of Wild-type (WT) or R1629Q Na_v1.5 channel and hβ1 subunit were achieved in human embryonic kidney cells by transient transfection. Sodium currents were recorded using whole cell patch-clamp protocols. No significant changes between WT and R1629Q currents were observed in current density or steady-state activation. However, hyperpolarized shift of steady–state inactivation curve was identified in cells expressing R1629Q channel (WT: V_1/2 = -81.1 ± 1.3 mV, n = 13; R1629Q: V_1/2 = -101.7 ± 1.2 mV, n = 18). Moreover, R1629Q channel showed enhanced intermediate inactivation and prolonged recovery time from inactivation. In summary, this study reveals that R1629Q mutation causes a distinct loss-of-function of the channel due to alter its electrophysiological characteristics, and facilitates our understanding of biophysical mechanisms of BrS.     

Thapsigargin selectively rescues the trafficking defective LQT2 channels G601S and F805C

Several mutations in the human ether-a-go-go-related K+ channel gene (HERG or KCNH2) cause long QT syndrome (LQT2) by reducing the intracellular transport (trafficking) of the channel protein to the cell surface. Drugs that bind to and block HERG channels (i.e. E4031) rescue the surface expression of some trafficking defective LQT2 mutations. Because these drugs potently block HERG current, their ability to correct congenital LQT is confounded by their risk of causing acquired LQT. We tested the hypothesis that pharmacological rescue can occur without HERG channel block. Thapsigargin (1 microM), a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor, rescued the surface expression of G601S, and it did so without blocking current. Thapsigargin-induced rescue and E4031-induced rescue caused complex glycosylation that was evident within 3 h of drug exposure. Disruption of the Golgi apparatus with brefeldin A prevented thapsigargin- and E4031-induced rescue of IG01S. Confocal imaging showed that G601S protein is predominantly "trapped" intracellularly and that both thapsigargin and E4031 promote its relocation to the surface membrane. We also studied two other trafficking defective LQT2 mutations. Thapsigargin rescued the C terminus mutation F805C but not N470D, whereas E4031 rescued N470D but not F805C. Other sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitors did not rescue G601S or F805C. This study 1) supports the hypothesis that the LQT2 trafficking defective phenotype can be reversed without blocking the channel; 2) demonstrates pharmacological rescue of a C terminus LQT2 mutation; and 3) shows that thapsigargin can correct trafficking defective phenotypes in more than one channel type and disease (i.e. LQT2 and cystic fibrosis).     

Targeting KV channels rescues retinal ganglion cells in vivo directly and by reducing inflammation

Retinal ganglion cell (RGC) degeneration is an important cause of visual impairment, and results in part from microglia-mediated inflammation. Numerous experimental studies have focused on identifying drug targets to rescue these neurons. We recently showed that K_V1.1 and K_V1.3 channels are expressed in adult rat RGCs and that siRNA -mediated knockdown of either channel reduces RGC death after optic nerve transection. Earlier we found that K_V1.3 channels also contribute to microglial activation and neurotoxicity; raising the possibility that these channels contribute to neurodegeneration through direct roles in RGCs and through inflammatory mechanisms. Here, RGC survival was increased by combined siRNA-mediated knockdown of K_V1.1 and K_V1.3 in RGCs, but survival was much greater when knockdown of either channel was combined with intraocular injection of a K_V1.3 channel blocker (agitoxin-2 or margatoxin). After axotomy, increased expression of several inflammation-related molecules preceded RGC loss and, consistent with a dual mechanism, their expression was differentially affected when channel knockdown in RGCs was combined with K_V1.3 blocker injection. K_V1.3 blockers reduced activation of retinal microglia and their tight apposition along RGC axon fascicles after axotomy, but did not prevent their migration from the inner plexiform to the damaged ganglion cell layer. Expression of several growth factors increased after axotomy; and again, there were differences following blocker injection compared with RGC-selective channel knockdown. These results provide evidence that K_V1.3 channels play important roles in apoptotic degeneration of adult RGCs through cell-autonomous mechanisms mediated by channels in the neurons, and non-autonomous mechanisms mediated by microglia and inflammation.Key words: neurotrauma, axotomy, optic nerve transection, microglial activation, apoptosis, K_V1.1, K_V1.3, siRNA in vivo, agitoxin-2, margatoxin     

Febrile temperatures unmask biophysical defects in Nav1.1 epilepsy mutations supportive of seizure initiation

Generalized epilepsy with febrile seizures plus (GEFS+) is an early onset febrile epileptic syndrome with therapeutic responsive (a)febrile seizures continuing later in life. Dravet syndrome (DS) or severe myoclonic epilepsy of infancy has a complex phenotype including febrile generalized or hemiclonic convulsions before the age of 1, followed by intractable myoclonic, complex partial, or absence seizures. Both diseases can result from mutations in the Na_v1.1 sodium channel, and initially, seizures are typically triggered by fever. We previously characterized two Na_v1.1 mutants—R859H (GEFS+) and R865G (DS)—at room temperature and reported a mixture of biophysical gating defects that could not easily predict the phenotype presentation as either GEFS+ or DS. In this study, we extend the characterization of Na_v1.1 wild-type, R859H, and R865G channels to physiological (37°C) and febrile (40°C) temperatures. At physiological temperature, a variety of biophysical defects were detected in both mutants, including a hyperpolarized shift in the voltage dependence of activation and a delayed recovery from fast and slow inactivation. Interestingly, at 40°C we also detected additional gating defects for both R859H and R865G mutants. The GEFS+ mutant R859H showed a loss of function in the voltage dependence of inactivation and an increased channel use-dependency at 40°C with no reduction in peak current density. The DS mutant R865G exhibited reduced peak sodium currents, enhanced entry into slow inactivation, and increased use-dependency at 40°C. Our results suggest that fever-induced temperatures exacerbate the gating defects of R859H or R865G mutants and may predispose mutation carriers to febrile seizures.     

Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Nav1.5

Background

The cardiac sodium channel Na_v1.5 is essential for the physiological function of the heart and causes cardiac arrhythmias and sudden death when mutated. Many disease-causing mutations in Na_v1.5 cause defects in protein trafficking, a cellular process critical to the targeting of Na_v1.5 to cell surface. However, the molecular mechanisms underlying the trafficking of Na_v1.5, in particular, the exit from the endoplasmic reticulum (ER) for cell surface trafficking, remain poorly understood.

Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation sites

Phosphorylation of the voltage-gated Na⁺ (Na_V) channel Na_V1.5 regulates cardiac excitability, yet the phosphorylation sites regulating its function and the underlying mechanisms remain largely unknown. Using a systematic, quantitative phosphoproteomic approach, we analyzed Na_V1.5 channel complexes purified from nonfailing and failing mouse left ventricles, and we identified 42 phosphorylation sites on Na_V1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic Na_V1.5 mutants revealed the roles of three phosphosites in regulating Na_V1.5 channel expression and gating. The phosphorylated serines S664 and S667 regulate the voltage dependence of channel activation in a cumulative manner, whereas the nearby S671, the phosphorylation of which is increased in failing hearts, regulates cell surface Na_V1.5 expression and peak Na⁺ current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, our results demonstrate that ventricular Na_V1.5 is highly phosphorylated and that the phosphorylation-dependent regulation of Na_V1.5 channels is highly complex, site specific, and dynamic.     

Characterization and Functional Restoration of a Potassium Channel Kir6.2 Pore Mutation Identified in Congenital Hyperinsulinism

The inwardly rectifying potassium channel Kir6.2 assembles with sulfonylurea receptor 1 to form the ATP-sensitive potassium (K_ATP) channels that regulate insulin secretion in pancreatic β-cells. Mutations in K_ATP channels underlie insulin secretion disease. Here, we report the characterization of a heterozygous missense Kir6.2 mutation, G156R, identified in congenital hyperinsulinism. Homomeric mutant channels reconstituted in COS cells show similar surface expression as wild-type channels but fail to conduct potassium currents. The mutated glycine is in the pore-lining transmembrane helix of Kir6.2; an equivalent glycine in other potassium channels has been proposed to serve as a hinge to allow helix bending during gating. We found that mutation of an adjacent asparagine, Asn-160, to aspartate, which converts the channel from a weak to a strong inward rectifier, on the G156R background restored ion conduction in the mutant channel. Unlike N160D channels, however, G156R/N160D channels are not blocked by intracellular polyamines at positive membrane potential and exhibit wild-type-like nucleotide sensitivities, suggesting the aspartate introduced at position 160 interacts with arginine at 156 to restore ion conduction and gating. Using tandem Kir6.2 tetramers containing G156R and/or N160D in designated positions, we show that one mutant subunit in the tetramer is insufficient to abolish conductance and that G156R and N160D can interact in the same or adjacent subunits to restore conduction. We conclude that the glycine at 156 is not essential for K_ATP channel gating and that the Kir6.2 gating defect caused by the G156R mutation could be rescued by manipulating chemical interactions between pore residues.     

Probing conformational rescue induced by a chemical corrector of F508del-cystic fibrosis transmembrane conductance regulator (CFTR) mutant

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that cause loss of function of the CFTR channel on the apical surface of epithelial cells. The major CF-causing mutation, F508del-CFTR, is misfolded, retained in the endoplasmic reticulum, and degraded. Small molecule corrector compounds have been identified using high throughput screens, which partially rescue the trafficking defect of F508del-CFTR, allowing a fraction of the mutant protein to escape endoplasmic reticulum retention and traffic to the plasma membrane, where it exhibits partial function as a cAMP-regulated chloride channel. A subset of such corrector compounds binds directly to the mutant protein, prompting the hypothesis that they rescue the biosynthetic defect by inducing improved protein conformation. We tested this hypothesis directly by evaluating the consequences of a corrector compound on the conformation of each nucleotide binding domain (NBD) in the context of the full-length mutant protein in limited proteolytic digest studies. Interestingly, we found that VRT-325 was capable of partially restoring compactness in NBD1. However, VRT-325 had no detectable effect on the conformation of the second half of the molecule. In comparison, ablation of the di-arginine sequence, R(553)XR(555) (F508del-KXK-CFTR), modified protease susceptibility of NBD1, NBD2, and the full-length protein. Singly, each intervention led to a partial correction of the processing defect. Together, these interventions restored processing of F508del-CFTR to near wild type. Importantly, however, a defect in NBD1 conformation persisted, as did a defect in channel activation after the combined interventions. Importantly, this defect in channel activation can be fully corrected by the addition of the potentiator, VX-770.