Ancillary Subunits Research Articles

Sex specific association of potassium channel subunits

Sex specific association of potassium channel subunits

Sexual dimorphism and oestrogen regulation of KCNE3 expression modulates the functional properties of KCNQ1 K+ channels

The KCNQ1 potassium channel associates with various KCNE ancillary subunits that drastically affect channel gating and pharmacology. Co-assembly with KCNE3 produces a current with nearly instantaneous activation, some time-dependent activation at very positive potentials, a linear current-voltage relationship and a 10-fold higher sensitivity to chromanol 293B. KCNQ1:KCNE3 channels are expressed in colonic crypts and mediate basolateral K(+) recycling required for Cl(-) secretion. We have previously reported the female-specific anti-secretory effects of oestrogen via KCNQ1:KCNE3 channel inhibition in colonic crypts. This study was designed to determine whether sex and oestrogen regulate the expression and function of KCNQ1 and KCNE3 in rat distal colon. Colonic crypts were isolated from Sprague-Dawley rats and used for whole-cell patch-clamp and to extract total RNA and protein. Sheets of epithelium were used for short-circuit current recordings. KCNE1 and KCNE3 mRNA and protein abundance were significantly higher in male than female crypts. No expression of KCNE2 was found and no difference was observed in KCNQ1 expression between male and female (at oestrus) colonic crypts. Male crypts showed a 2.2-fold higher level of association of KCNQ1 and KCNE3 compared to female cells. In female colonic crypts, KCNQ1 and KCNE3 protein expression fluctuated throughout the oestrous cycle and 17β-oestradiol (E2 10 nM) produced a rapid (<15 min) dissociation of KCNQ1 and KCNE3 in female crypts only. Whole-cell K(+) currents showed a linear current-voltage relationship in male crypts, while K(+) currents in colonic crypts isolated from females displayed voltage-dependent outward rectification. Currents in isolated male crypts and epithelial sheets were 10-fold more sensitive to specific KCNQ1 inhibitors, such as chromanol 293B and HMR-1556, than in female. The effect of E2 on K(+) currents mediated by KCNQ1 with or without different β-subunits was assayed from current-voltage relations elicited in CHO cells transfected with KCNQ1 and KCNE3 or KCNE1 cDNA. E2 (100 nM) reduced the currents mediated by the KCNQ1:KCNE3 potassium channel and had no effect on currents via KCNQ1:KCNE1 or KCNQ1 alone. Currents mediated by the complex formed by KCNQ1 and the mutant KCNE3-S82A β-subunit (mutation of the site for PKCδ-promoted phosphorylation and modulation of the activity of KCNE3) showed rapid run-down and insensitivity to E2. Together, these data suggest that oestrogen regulates the expression of the KCNE1 and KCNE3 and with it the gating and pharmacological properties of the K(+) conductance required for Cl(-) secretion. The decreased association of the KCNQ1:KCNE3 channel complex promoted by oestrogen exposure underlies the molecular mechanism for the sexual dimorphism and oestrous cycle dependence of the anti-secretory actions of oestrogen in the intestine.

KCNE1 and KCNE2 Provide a Checkpoint Governing Voltage-Gated Potassium Channel α-Subunit Composition

Voltage-gated potassium (Kv) currents generated by N-type α-subunit homotetramers inactivate rapidly because an N-terminal ball domain blocks the channel pore after activation. Hence, the inactivation rate of heterotetrameric channels comprising both N-type and non-N-type (delayed rectifier) α-subunits depends upon the number of N-type α-subunits in the complex. As Kv channel inactivation and inactivation recovery rates regulate cellular excitability, the composition and expression of these heterotetrameric complexes are expected to be tightly regulated. In a companion article, we showed that the single transmembrane segment ancillary (β) subunits KCNE1 and KCNE2 suppress currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels, by trapping them early in the secretory pathway. Here, we show that this trapping is prevented by coassembly of the N-type α-subunits with intra-subfamily delayed rectifier α-subunits. Extra-subfamily delayed rectifier α-subunits, regardless of their capacity to interact with KCNE1 and KCNE2, cannot rescue Kv1.4 or Kv3.4 surface expression unless engineered to interact with them using N-terminal A and B domain swapping. The KCNE1/2-enforced checkpoint ensures N-type α-subunits only reach the cell surface as part of intra-subfamily mixed-α complexes, thereby governing channel composition, inactivation rate, and—by extension—cellular excitability.

KCNE1 and KCNE2 Inhibit Forward Trafficking of Homomeric N-Type Voltage-Gated Potassium Channels

Potassium currents generated by voltage-gated potassium (Kv) channels comprising α-subunits from the Kv1, 2, and 3 subfamilies facilitate high-frequency firing of mammalian neurons. Within these subfamilies, only three α-subunits (Kv1.4, Kv3.3, and Kv3.4) generate currents that decay rapidly in the open state because an N-terminal ball domain blocks the channel pore after activation—a process termed N-type inactivation. Despite its importance to shaping cellular excitability, little is known of the processes regulating surface expression of N-type α-subunits, versus their slowly inactivating (delayed rectifier) counterparts. Here we found that currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels are all strongly suppressed by the single transmembrane domain ancillary (β) subunits KCNE1 and KCNE2. A combination of electrophysiological, biochemical, and immunofluorescence analyses revealed this suppression is due to KCNE1 and KCNE2 retaining Kv1.4 and Kv3.4 intracellularly, early in the secretory pathway. The retention is specific, requires α-β coassembly, and does not involve the dynamin-dependent endocytosis pathway. However, the small fraction of Kv3.4 that escapes KCNE-dependent retention is regulated by dynamin-dependent endocytosis. The findings illustrate two contrasting mechanisms controlling surface expression of N-type Kv α-subunits and therefore, potentially, cellular excitability and refractory periods.

Role of BK channels in hypertension and potassium secretion

This review summarizes recent studies of hypertension associated with a defect in renal K excretion due to genetic deletions of various components of the large, Ca-activated K channel (BK), and describes new evidence and theories regarding K secretory roles of BK in intercalated cells. Isolated perfused tubule methods have revealed the importance of BK in flow-induced K secretion. Subsequently, mice with genetically deleted BK subunits revealed the complexities of BK-mediated K secretion. Deletion of BKα results in extreme aldosteronism, hypertension, and an absence of flow-induced K secretion. Deletion of the BKβ1 ancillary subunit results in decreased handling of a K load, increased plasma K, mild aldosteronism and hypertension that is exacerbated by a high K diet. Deletion of BKβ4 (β4KO) leads to insufficient K handling, high plasma K, fluid retention, but with milder hypertension. Fluid retention in β4KO may be the result of insufficient flow-induced secretion of adenosine triphosphate (ATP), which normally inhibits epithelial Na channels (ENaCs). Classical physiological analysis of electrolyte handling in knockout mice has enlightened our understanding of the mechanism of handling K loads by renal K channels. Studies have focused on the different roles of BK-α/β1 and BK-α/β4 in the kidney. BKβ1 hypertension may be a 'three-hit' hypertension, involving a K secretory defect, elevated production of aldosterone, and increased vascular tone. The disorders observed in BK knockout mice have shed new insights on the importance of proper renal K handling for maintaining volume balance and blood pressure.

Protein Kinase C Influence on Kv4.3 Closed State Inactivation

Kv4.3 is expressed as a short and a long form that differ by a 19 amino acid insertion downstream from the S6 transmembrane segment. It is typically expressed with ancillary subunits, the most prominent of which are cytoplasmic proteins called KChIPs. Activation of PKC by phorbol esters modulates closed-state inactivation (CSI) in Kv4.3. PKC reduces CSI in the short form of Kv4.3 (Kv4.3S), however, the 19 amino acid insertion in Kv4.3L changes the effect to increasing the magnitude of CSI through phosphorylation of a threonine unique to the long form. KChIP2b blocks the effect of PKC on CSI in both Kv4.3S and Kv4.3L. In contrast, 70-amino acid KChIP2d was permissive for PKC modulation of CSI characteristic seen in both forms of Kv4.3. These data suggested the presence of additional PKC phosphorylation sites in Kv4.3. Bioinformatic analysis suggested that the most likely PKC phosphorylation sites were in the N-terminal cytoplasmic domain of Kv4.3 at S47, T53, T100 (all in the T1 domain), and S165 (in the T1-S1 linker). Mutagenesis of any of the four amino acids reduced CSI; T53, T100, or S165 were influenced by PKC to a much lesser extent than WT Kv4.3S, while S47A dramatically reduced CSI. In all cases, there was little difference between mutations in the short and long Kv4.3 isoforms. Mutations in S6 at amino acids V399 and V401 have been shown to have dramatic effects on CSI. In the presence of these mutations, the Kv4.3L insert has no influence on CSI upon PKC induction. These data suggest that the isoform-specific modulation of CSI by PKC in Kv4.3 involves complex interactions of the cytoplasmic N and C termini of the channels, and that the phosphorylation of the long form acts by separate and perhaps unique mechanism.

KCNQ1 Channels Voltage Dependence through a Voltage-dependent Binding of the S4-S5 Linker to the Pore Domain

Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1-S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5-S6) is surrounded by four voltage sensor domains (S1-S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5(L)) and S6 C terminus (S6(T)). From these data, we hypothesized that S4S5(L) behaves like a ligand specifically interacting with S6(T) and stabilizing the closed state. To test this hypothesis, we designed plasmid-encoded peptides corresponding to portions of S4S5(L) and S6(T) of the voltage-gated potassium channel KCNQ1 and evaluated their effects on the channel activity in the presence and absence of the ancillary subunit KCNE1. We showed that S4S5(L) peptides inhibit KCNQ1, in a reversible and state-dependent manner. S4S5(L) peptides also inhibited a voltage-independent KCNQ1 mutant. This inhibition was competitively prevented by a peptide mimicking S6(T), consistent with S4S5(L) binding to S6(T). Val(254) in S4S5(L) is known to contact Leu(353) in S6(T) when the channel is closed, and mutations of these residues alter the coupling between the two regions. The same mutations introduced in peptides altered their effects, further confirming S4S5(L) binding to S6(T). Our results suggest a mechanistic model in which S4S5(L) acts as a voltage-dependent ligand bound to its receptor on S6 at rest. This interaction locks the channel in a closed state. Upon plasma membrane depolarization, S4 pulls S4S5(L) away from S6(T), allowing channel opening.

Expression and distribution of voltage-gated ion channels in ferret sinoatrial node

Spontaneous diastolic depolarization in the sinoatrial (SA) node enables it to serve as pacemaker of the heart. The variable cell morphology within the SA node predicts that ion channel expression would be heterogeneous and different from that in the atrium. To evaluate ion channel heterogeneity within the SA node, we used fluorescent in situ hybridization to examine ion channel expression in the ferret SA node region and atrial appendage. SA nodal cells were distinguished from surrounding cardiac myocytes by expression of the slow (SA node) and cardiac (surrounding tissue) forms of troponin I. Nerve cells in the sections were identified by detection of GAP-43 and cytoskeletal middle neurofilament. Transcript expression was characterized for the 4 hyperpolarization-activated cation channels, 6 voltage-gated Na(+) channels, 3 voltage-gated Ca(2+) channels, 24 voltage-gated K(+) channel α-subunits, and 3 ancillary subunits. To ensure that transcript expression was representative of protein expression, immunofluorescence was used to verify localization patterns of voltage-dependent K(+) channels. Colocalizations were performed to observe any preferential patterns. Some overlapping and nonoverlapping binding patterns were observed. Measurement of different cation channel transcripts showed heterogeneous expression with many different patterns of expression, attesting to the complexity of electrical activity in the SA node. This study provides insight into the possible role ion channel heterogeneity plays in SA node pacemaker activity.

Synchrotron radiation circular dichroism spectroscopy-defined structure of the C-terminal domain of NaChBac and its role in channel assembly

Extramembranous domains play important roles in the structure and function of membrane proteins, contributing to protein stability, forming association domains, and binding ancillary subunits and ligands. However, these domains are generally flexible, making them difficult or unsuitable targets for obtaining high-resolution X-ray and NMR structural information. In this study we show that the highly sensitive method of synchrotron radiation circular dichroism (SRCD) spectroscopy can be used as a powerful tool to investigate the structure of the extramembranous C-terminal domain (CTD) of the prokaryotic voltage-gated sodium channel (Na(V)) from Bacillus halodurans, NaChBac. Sequence analyses predict its CTD will consist of an unordered region followed by an alpha-helix, which has a propensity to form a multimeric coiled-coil motif, and which could form an association domain in the homotetrameric NaChBac channel. By creating a number of shortened constructs we have shown experimentally that the CTD does indeed contain a stretch of approximately 20 alpha-helical residues preceded by a nonhelical region adjacent to the final transmembrane segment and that the efficiency of assembly of channels in the membrane progressively decreases as the CTD residues are removed. Analyses of the CTDs of 32 putative prokaryotic Na(V) sequences suggest that a CTD helical bundle is a structural feature conserved throughout the bacterial sodium channel family.

Kv1.3 is the exclusive voltage‐gated K+ channel of platelets and megakaryocytes: roles in membrane potential, Ca2+ signalling and platelet count

A delayed rectifier voltage-gated K+ channel (Kv) represents the largest ionic conductance of platelets and megakaryocytes, but is undefined at the molecular level. Quantitative RT-PCR of all known Kv α and ancillary subunits showed that only Kv1.3 (KCNA3) is substantially expressed in human platelets. Furthermore, megakaryocytes from Kv1.3−/− mice or from wild-type mice exposed to the Kv1.3 blocker margatoxin completely lacked Kv currents and displayed substantially depolarised resting membrane potentials. In human platelets, margatoxin reduced the P2X1- and thromboxaneA2 receptor-evoked [Ca2+]i increases and delayed the onset of store-operated Ca2+ influx. Megakaryocyte development was normal in Kv1.3−/− mice, but the platelet count was increased, consistent with a role of Kv1.3 in apoptosis or decreased platelet activation. We conclude that Kv1.3 forms the Kv channel of the platelet and megakaryocyte, which sets the resting membrane potential, regulates agonist-evoked Ca2+ increases and influences circulating platelet numbers.

Ancillary Subunits Associated With Voltage-Dependent K+Channels

Since the first discovery of Kvbeta-subunits more than 15 years ago, many more ancillary Kv channel subunits were characterized, for example, KChIPs, KCNEs, and BKbeta-subunits. The ancillary subunits are often integral parts of native Kv channels, which, therefore, are mostly multiprotein complexes composed of voltage-sensing and pore-forming Kvalpha-subunits and of ancillary or beta-subunits. Apparently, Kv channels need the ancillary subunits to fulfill their many different cell physiological roles. This is reflected by the large structural diversity observed with ancillary subunit structures. They range from proteins with transmembrane segments and extracellular domains to purely cytoplasmic proteins. Ancillary subunits modulate Kv channel gating but can also have a great impact on channel assembly, on channel trafficking to and from the cellular surface, and on targeting Kv channels to different cellular compartments. The importance of the role of accessory subunits is further emphasized by the number of mutations that are associated in both humans and animals with diseases like hypertension, epilepsy, arrhythmogenesis, periodic paralysis, and hypothyroidism. Interestingly, several ancillary subunits have in vitro enzymatic activity; for example, Kvbeta-subunits are oxidoreductases, or modulate enzymatic activity, i.e., KChIP3 modulates presenilin activity. Thus different modes of beta-subunit association and of functional impact on Kv channels can be delineated, making it difficult to extract common principles underlying Kvalpha- and beta-subunit interactions. We critically review present knowledge on the physiological role of ancillary Kv channel subunits and their effects on Kv channel properties.

Ancillary Subunits Regulate PKC Mediated Effects on Closed-State Inactivation of Kv4.3

Kv4 channels are expressed with a variety of ancillary subunits in vivo. The most prominent of these proteins are the KChIPs, a family of cytoplasmic proteins that modulate channel gating and act as chaperones. We have previously shown that heterologously expressed Kv4.3 is regulated by PKC. After induction of PKC by PMA, current expression level is reduced. PKC also influences closed-state inactivation (CSI) in an isoform dependent manner; CSI is decreased upon PKC induction in Kv4.3-S, and increased in PKC-L. To understand the role of PKC on modulation of Kv4-based currents, we expressed channels in the presence of three KChIP2 isoforms and compared their responses to PKC. Two KChIP2 isoforms, KChIP2a and 2b, negated PKC influence on channel gating, kinetics, and expression levels in both Kv4.3-S and Kv4.3-L. In contrast, 70-amino acid KChIP2d was permissive for PKC modulation with respect to both CSI and current expression, while allowing the fast recovery from open-state inactivation characteristic of other KChIP2 isoforms. Additionally, the KChIP2d effects on CSI in Kv4.3-L were dependent on the presence of a putative PKC phosphorylation site in the C terminus. These data suggest a different physiological role for KChIP2d than the other KChIP2 isoforms, and suggest that the longer forms of KChIP2 interact with the regions of Kv4.3 affected by PKC, while KChIP2d interacts with the channel in a manner that allows PKC modulation while still accelerating recovery.

KCNQ1 K+ Channels are Involved in Lipopolysaccharide-induced Apoptosis of Distal Kidney Cells

Most bacteria initiate host inflammatory responses through interactions with epithelial cells. Lipopolysaccharide (LPS), a component of the bacterial cell wall is a major cause of septic shock in emergency care units and in the pathogenesis of acute renal failure. Kidney cells exposed to LPS undergo apoptotic changes, including cell volume decrease, phosphatidylserine exposure, caspase-3- and membrane K⁺ conductance -activation. Whole-cell configuration was used to identify K⁺ channels in primary and immortalized culture of mice distal convoluted tubules. LPS exposure induced a 3 fold increase in intracellular cAMP concentration and the activation of an outwardly rectifying K⁺ conductance in both immortalized and primary culture of distal cells. This LPS-induced current exhibited KCNQ1 K⁺ channel characteristics, i.e. inhibition by quinidine, chromanol293B and low dose of HMR1556 (IC50<1 µM) and insensitive to TEA and charybdotoxin. The background-like biophysical properties of the current suggest that the KCNQ1 pore-forming subunit is associated with a KCNE2 or KCNE3 ancillary subunit. RT-PCR experiments confirmed the presence of KCNQ1 and KCNE3 mRNA transcripts in primary culture of distal segments. Activation of the KCNQ1/KCNE3 K⁺ current appeared to be an essential step in the LPS-induced apoptosis process since HMR1556 blocked the LPS-induced- cell volume decrease, -caspase-3 activation and -phosphatidylserine exposure.

Role of voltage-gated calcium channels in epilepsy

It is well established that idiopathic generalized epilepsies (IGEs) show a polygenic origin and may arise from dysfunction of various types of voltage- and ligand-gated ion channels. There is an increasing body of literature implicating both high- and low-voltage-activated (HVA and LVA) calcium channels and their ancillary subunits in IGEs. Cav2.1 (P/Q-type) calcium channels control synaptic transmission at presynaptic nerve terminals, and mutations in the gene encoding the Cav2.1 alpha1 subunit (CACNA1A) have been linked to absence seizures in both humans and rodents. Similarly, mutations and loss of function mutations in ancillary HVA calcium channel subunits known to co-assemble with Cav2.1 result in IGE phenotypes in mice. It is important to note that in all these mouse models with mutations in HVA subunits, there is a compensatory increase in thalamic LVA currents which likely leads to the seizure phenotype. In fact, gain-of-function mutations have been identified in Cav3.2 (an LVA or T-type calcium channel encoded by the CACNA1H gene) in patients with congenital forms of IGEs, consistent with increased excitability of neurons as a result of enhanced T-type channel function. In this paper, we provide a broad overview of the roles of voltage-gated calcium channels, their mutations, and how they might contribute to the river that terminates in epilepsy.

KCNE4 suppresses Kv1.3 currents by modulating trafficking, surface expression and channel gating

Voltage-dependent potassium channels (Kv) play a crucial role in the activation and proliferation of leukocytes. Kv channels are either homo- or hetero-oligomers. This composition modulates their surface expression and serves as a mechanism for regulating channel activity. Kv channel interaction with accessory subunits provides mechanisms for channels to respond to stimuli beyond changes in membrane potential. Here, we demonstrate that KCNE4 (potassium voltage-gated channel subfamily E member 4), but not KCNE2, functions as an inhibitory Kv1.3 partner in leukocytes. Kv1.3 trafficking, targeting and activity are altered by the presence of KCNE4. KCNE4 decreases current density, slows activation, accelerates inactivation, increases cumulative inactivation, retains Kv1.3 in the ER and impairs channel targeting to lipid raft microdomains. KCNE4 associates with Kv1.3 in the ER and decreases the number of Kv1.3 channels at the cell surface, which diminishes cell excitability. Kv1.3 and KCNE4 are differentially regulated upon activation or immunosuppression in macrophages. Thus, lipopolysaccharide-induced activation increases Kv1.3 and KCNE4 mRNA, whereas dexamethasone triggers a decrease in Kv1.3 with no changes in KCNE4. The channelosome composition determines the activity and affects surface expression and membrane localization. Therefore, KCNE4 association might play a crucial role in controlling immunological responses. Our results indicate that KCNE ancillary subunits could be new targets for immunomodulation.

Augmented potassium current is a shared phenotype for two genetic defects associated with familial atrial fibrillation

Mutations in multiple genes have been implicated in familial atrial fibrillation (AF), but the underlying mechanisms, and thus implications for therapy, remain ill-defined. Among 231 participants in the Vanderbilt AF Registry, we found a mutation in KCNQ1 (encoding the α-subunit of slow delayed rectifier potassium current [ I Ks]) and separately a mutation in natriuretic peptide precursor A ( NPPA) gene (encoding atrial natriuretic peptide, ANP), both segregating with early onset lone AF in different kindreds. The functional effects of these mutations yielded strikingly similar I Ks “gain-of-function.” In Chinese Hamster Ovary (CHO) cells, coexpression of mutant KCNQ1 with its ancillary subunit KCNE1 generated ∼ 3-fold larger currents that activated much faster than wild-type (WT)- I Ks. Application of the WT NPPA peptide fragment produced similar changes in WT- I Ks, and these were exaggerated with the mutant NPPA S64R peptide fragment. Anantin, a competitive ANP receptor antagonist, completely inhibited the changes in I Ks gating observed with NPPA S64R. Computational simulations identified accelerated transitions into open states as the mechanism for variant I Ks gating. Incorporating these I Ks changes into computed human atrial action potentials (AP) resulted in 37% shortening (120 vs. 192 ms at 300 ms cycle length), reflecting loss of the phase II dome which is dependent on L-type calcium channel current. We found striking functional similarities due to mutations in KCNQ1 and NPPA genes which led to I Ks “gain-of-function”, atrial AP shortening, and consequently altered calcium current as a common mechanism between diverse familial AF syndromes.

Hypertension of Kcnmb1 −/− is linked to deficient K secretion and aldosteronism

Mice lacking the beta1-subunit (gene, Kcnmb1; protein, BK-beta1) of the large Ca-activated K channel (BK) are hypertensive. This phenotype is thought to result from diminished BK currents in vascular smooth muscle where BK-beta1 is an ancillary subunit. However, the beta1-subunit is also expressed in the renal connecting tubule (CNT), a segment of the aldosterone-sensitive distal nephron, where it associates with BK and facilitates K secretion. Because of the correlation between certain forms of hypertension and renal defects, particularly in the distal nephron, it was determined whether the hypertension of Kcnmb1(-/-) has a renal origin. We found that Kcnmb1(-/-) are hypertensive, volume expanded, and have reduced urinary K and Na clearances. These conditions are exacerbated when the animals are fed a high K diet (5% K; HK). Supplementing HK-fed Kcnmb1(-/-) with eplerenone (mineralocorticoid receptor antagonist) corrected the fluid imbalance and more than 70% of the hypertension. Finally, plasma [aldo] was elevated in Kcnmb1(-/-) under basal conditions (control diet, 0.6% K) and increased significantly more than wild type when fed the HK diet. We conclude that the majority of the hypertension of Kcnmb1(-/-) is due to aldosteronism, resulting from renal potassium retention and hyperkalemia.

KTN (RCK) Domains Regulate K+ Channels and Transporters by Controlling the Dimer-Hinge Conformation

SummaryKTN (RCK) domains are nucleotide-binding folds that form the cytoplasmic regulatory complexes of various K+ channels and transporters. The mechanisms these proteins use to control their transmembrane pore-forming counterparts remains unclear despite numerous electrophysiological and structural studies. KTN (RCK) domains consistently crystallize as dimers within the asymmetric unit, forming a pronounced hinge between two Rossmann folds. We have previously proposed that modification of the hinge angle plays an important role in activating the associated membrane-integrated components of the channel or transporter. Here we report the structure of the C-terminal, KTN-bearing domain of the E. coli KefC K+ efflux system in association with the ancillary subunit, KefF, which is known to stabilize the conductive state. The structure of the complex and functional analysis of KefC variants reveal that control of the conformational flexibility inherent in the KTN dimer hinge is modulated by KefF and essential for regulation of KefC ion flux.

NovelKCNE3mutation reduces repolarizing potassium current and associated with long QT syndrome

Long QT syndrome (LQTS) is an inherited disease involving mutations in the genes encoding a number of cardiac ion channels and a membrane adaptor protein. Among the genes that are responsible for LQTS, KCNE1 and KCNE2 are members of the KCNE family of genes, and function as ancillary subunits of Kv channels. The third KCNE gene, KCNE3, is expressed in cardiac myocytes and interacts with KCNQ1 to change the channel properties. However, KCNE3 has never been linked to LQTS. To investigate the association between KCNE3 and LQTS, we conducted a genetic screening of KCNE3 mutations and single nucleotide polymorphisms (SNPs) in 485 Japanese LQTS probands using DHPLC-WAVE system and direct sequencing. Consequently, we identified two KCNE3 missense mutations, located in the N- and C-terminal domains. The functional effects of these mutations were examined by heterologous expression systems using CHO cells stably expressing KCNQ1. One mutation, p.R99lambdaH was identified in a 76-year-old woman who suffered torsades de pointes (TdP) after administration of disopyramide. Another mutation, p.T4A was identified in a 16-year-old boy and 67-year-old woman. Although the boy carried another KCNH2 mutation, he was asymptomatic. On the other hand, the woman suffered from hypokalemia-induced TdP. In a series of electrophysiological analyses, the KCNQ1(Q1)+KCNE3(E3)-R99lambdaH channel significantly reduced outward current compared to Q1+E3-WT, though the current density of the Q1+E3-T4A channel displayed no statistical significance. This is the first report of KCNE3 mutations associated with LQTS. Screening for variants in the KCNE3 gene is of clinical importance for LQTS patients.

HERG biosynthesis: the positive influence of negative charge

HERG biosynthesis: the positive influence of negative charge