The Role of the Carboxyl Terminus Helix C-D Linker in Regulating KCNQ3 K+ Current Amplitudes by Controlling Channel Trafficking

In the central and peripheral nervous system, the assembly of KCNQ3 with KCNQ2 as mostly heteromers, but also homomers, underlies “M-type” currents, a slowly-activating voltage-gated K⁺ current that plays a dominant role in neuronal excitability. KCNQ3 homomers yield much smaller currents compared to KCNQ2 or KCNQ4 homomers and KCNQ2/3 heteromers. This smaller current has been suggested to result either from divergent channel surface expression or from a pore that is more unstable in KCNQ3. Channel surface expression has been shown to be governed by the distal part of the C-terminus in which helices C and D are critical for channel trafficking and assembly. A sequence alignment of this region in KCNQ channels shows that KCNQ3 possesses a longer linker between helix C and D compared to the other KCNQ subunits. Here, we investigate the role of the extra residues of this linker on KCNQ channel expression. Deletion of these residues increased KCNQ3 current amplitudes. Total internal reflection fluorescence imaging and plasma membrane protein assays suggest that the increase in current is due to a higher surface expression of the channels. Conversely, introduction of the extra residues into the linker between helices C and D of KCNQ4 reduced current amplitudes by decreasing the number of KCNQ4 channels at the plasma membrane. Confocal imaging suggests a higher fraction of channels, which possess the extra residues of helix C-D linker, were retained within the endoplasmic reticulum. Such retention does not appear to lead to protein accumulation and activation of the unfolded protein response that regulates protein folding and maintains endoplasmic reticulum homeostasis. Taken together, we conclude that extra helix C-D linker residues play a role in KCNQ3 current amplitudes by controlling the exit of the channel from the endoplasmic reticulum.     

Role of calmodulin in neuronal Kv7/KCNQ potassium channels and epilepsy

Neuronal Kv7/KCNQ channels are critical regulators of neuronal excitability since they potently suppress repetitive firing of action potentials. These voltage-dependent potassium channels are composed mostly of Kv7.2 / KCNQ2 and KvT.3 / KCNQ3 subunits that show overlapping distribution throughout the brain and in the peripheral nervous system. They are also called ＇M-channels＇ since their inhibition by muscarinic agonists leads to a profound increase in action potential firing. Consistent with their ability to suppress seizures and attenuate chronic inflammatory and neuropathic pain, mutations in the KCNQ2 and KCNQ3 genes are associated with benign familial neonatal convulsions, a dominantly-inherited epilepsy in infancy. Recently, de novo mutations in the KCNQ2 gene have been linked to early onset epileptic encephalopathy. Notably, some of these mutations are clustered in a region of the intracellular cytoplasmic tail of Kv7.2 that interacts with a ubiquitous calcium sensor, calmodulin. In this review, we highlight the recent advances in understanding the role of calmodulin in modulating physiological function of neuronal Kv7 channels including their biophysical properties, assembly, and trafficking. We also summarize recent studies that have investigated functional impact of epilepsy-associated mutations localized to the calmodulin binding domains of Kv7.2.     

A Change in Configuration of the Calmodulin-KCNQ Channel Complex Underlies Ca2+-Dependent Modulation of KCNQ Channel Activity

All subtypes of KCNQ channel subunits (KCNQ1-5) require calmodulin as a co-factor for functional channels. It has been demonstrated that calmodulin plays a critical role in KCNQ channel trafficking as well as calcium-mediated current modulation. However, how calcium-bound calmodulin suppresses the M-current is not well understood. In this study, we investigated the molecular mechanism of KCNQ2 current suppression mediated by calcium-bound calmodulin. We show that calcium induced slow calmodulin dissociation from the KCNQ2 channel subunit. In contrast, in homomeric KCNQ3 channels, calcium facilitated calmodulin binding. We demonstrate that this difference in calmodulin binding was due to the unique cysteine residue in the KCNQ2 subunit at aa 527 in Helix B, which corresponds to an arginine residue in other KCNQ subunits including KCNQ3. In addition, a KCNQ2 channel associated protein AKAP79/150 (79 for human, 150 for rodent orthologs) also preferentially bound calcium-bound calmodulin. Therefore, the KCNQ2 channel complex was able to retain calcium-bound calmodulin either through the AKPA79/150 or KCNQ3 subunit. Functionally, increasing intracellular calcium by ionomycin suppressed currents generated by KCNQ2, KCNQ2(C527R) or heteromeric KCNQ2/KCNQ3 channels to an equivalent extent. This suggests that a change in the binding configuration, rather than dissociation of calmodulin, is responsible for KCNQ current suppression. Furthermore, we demonstrate that KCNQ current suppression was accompanied by reduced KCNQ affinity toward phosphatidylinositol 4,5-bisphosphate (PIP2) when assessed by a voltage-sensitive phosphatase, Ci-VSP. These results suggest that a rise in intracellular calcium induces a change in the configuration of CaM-KCNQ binding, which leads to the reduction of KCNQ affinity for PIP2 and subsequent current suppression.     

Cdk-mediated phosphorylation of the Kvβ2 auxiliary subunit regulates Kv1 channel axonal targeting

Kv1 channels are concentrated at specific sites in the axonal membrane, where they regulate neuronal excitability. Establishing these distributions requires regulated dissociation of Kv1 channels from the neuronal trafficking machinery and their subsequent insertion into the axonal membrane. We find that the auxiliary Kvβ2 subunit of Kv1 channels purified from brain is phosphorylated on serine residues 9 and 31, and that cyclin-dependent kinase (Cdk)-mediated phosphorylation at these sites negatively regulates the interaction of Kvβ2 with the microtubule plus end-tracking protein EB1. Endogenous Cdks, EB1, and Kvβ2 phosphorylated at serine 31 are colocalized in the axons of cultured hippocampal neurons, with enrichment at the axon initial segment (AIS). Acute inhibition of Cdk activity leads to intracellular accumulation of EB1, Kvβ2, and Kv1 channel subunits within the AIS. These studies reveal a new regulatory mechanism for the targeting of Kv1 complexes to the axonal membrane through the reversible Cdk phosphorylation-dependent binding of Kvβ2 to EB1.     

Regulation of the voltage-gated K(+) channels KCNQ2/3 and KCNQ3/5 by ubiquitination. Novel role for Nedd4-2

The muscarine-sensitive K(+) current (M-current) stabilizes the resting membrane potential in neurons, thus limiting neuronal excitability. The M-current is mediated by heteromeric channels consisting of KCNQ3 subunits in association with either KCNQ2 or KCNQ5 subunits. The role of KCNQ2/3/5 in the regulation of neuronal excitability is well established; however, little is known about the mechanisms that regulate the cell surface expression of these channels. Ubiquitination by the Nedd4/Nedd4-2 ubiquitin ligases is known to regulate a number of membrane ion channels and transporters. In this study, we investigated whether Nedd4/Nedd4-2 could regulate KCNQ2/3/5 channels. We found that the amplitude of the K(+) currents mediated by KCNQ2/3 and KCNQ3/5 were reduced by Nedd4-2 (but not Nedd4) in a Xenopus oocyte expression system. Deletion experiments showed that the C-terminal region of the KCNQ3 subunit is required for the Nedd4-2-mediated regulation of the heteromeric channels. Glutathione S-transferase fusion pulldowns and co-immunoprecipitations demonstrated a direct interaction between KCNQ2/3 and Nedd4-2. Furthermore, Nedd4-2 could ubiquitinate KCNQ2/3 in transfected cells. Taken together, these data suggest that Nedd4-2 is potentially an important regulator of M-current activity in the nervous system.     

Made for "anchorin": Kv7.2/7.3 (KCNQ2/KCNQ3) channels and the modulation of neuronal excitability in vertebrate axons

Kv7.2 and Kv7.3 (encoded by KCNQ2 and KCNQ3) are homologous subunits forming a widely expressed neuronal voltage-gated K(+) (Kv) channel. Hypomorphic mutations in either KCNQ2 or KCNQ3 cause a highly penetrant, though transient, human phenotype-epilepsy during the first months of life. Some KCNQ2 mutations also cause involuntary muscle rippling, or myokymia, which is indicative of motoneuron axon hyperexcitability. Kv7.2 and Kv7.3 are concentrated at axonal initial segments (AISs), and at nodes of Ranvier in the central and peripheral nervous system. Kv7.2 and Kv7.3 share a novel ～80 residue C-terminal domain bearing an "anchor" motif, which interacts with ankyrin-G and is required for channel AIS (and likely, nodal) localization. This domain includes the sequence IAEGES/TDTD, which is analogous (not homologous) to the ankyrin-G interaction motif of voltage-gated Na(+) (Na(V)) channels. The KCNQ subfamily is evolutionarily ancient, with two genes (KCNQ1 and KCNQ5) persisting as orthologues in extant bilaterian animals from worm to man. However, KCNQ2 and KCNQ3 arose much more recently, in the interval between the divergence of extant jawless and jawed vertebrates. This is precisely the interval during which myelin and saltatory conduction evolved. The natural selection for KCNQ2 and KCNQ3 appears to hinge on these subunits' unique ability to be coordinately localized with Na(V) channels by ankyrin-G, and the resulting enhancement in the reliability of neuronal excitability.     

Some Pharmacological Properties Of Neural KCNQ Channels

The lecture summarizes data on the structural and pharmacological properties of KCNQ channels, in particular KCNQ2-5, associated primarily with the nervous system. The KCNQ channels are known to play a crucial role in the control of neuronal excitability. Two classes of drugs altering KCNQ channel activity and therefore producing considerable changes in neuronal excitability are described. These are KCNQ/M channel blocking agents (linopirdine, TEA) and KCNQ channel enhancers (retigabine).     

Drosophila KCNQ channel displays evolutionarily conserved electrophysiology and pharmacology with mammalian KCNQ channels

Of the five human KCNQ (Kv7) channels, KCNQ1 with auxiliary subunit KCNE1 mediates the native cardiac I(Ks) current with mutations causing short and long QT cardiac arrhythmias. KCNQ4 mutations cause deafness. KCNQ2/3 channels form the native M-current controlling excitability of most neurons, with mutations causing benign neonatal febrile convulsions. Drosophila contains a single KCNQ (dKCNQ) that appears to serve alone the functions of all the duplicated mammalian neuronal and cardiac KCNQ channels sharing roughly 50-60% amino acid identity therefore offering a route to investigate these channels. Current information about the functional properties of dKCNQ is lacking therefore we have investigated these properties here. Using whole cell patch clamp electrophysiology we compare the biophysical and pharmacological properties of dKCNQ with the mammalian neuronal and cardiac KCNQ channels expressed in HEK cells. We show that Drosophila KCNQ (dKCNQ) is a slowly activating and slowly-deactivating K(+) current open at sub-threshold potentials that has similar properties to neuronal KCNQ2/3 with some features of the cardiac KCNQ1/KCNE1 accompanied by conserved sensitivity to a number of clinically relevant KCNQ blockers (chromanol 293B, XE991, linopirdine) and opener (zinc pyrithione). We also investigate the molecular basis of the differential selectivity of KCNQ channels to the opener retigabine and show a single amino acid substitution (M217W) can confer sensitivity to dKCNQ. We show dKCNQ has similar electrophysiological and pharmacological properties as the mammalian KCNQ channels, allowing future study of physiological and pathological roles of KCNQ in Drosophila and whole organism screening for new modulators of KCNQ channelopathies.     

Surface expression and single channel properties of KCNQ2/KCNQ3, M-type K+ channels involved in epilepsy

Mutations in either KCNQ2 or KCNQ3 underlie benign familial neonatal convulsions (BFNC), an inherited epilepsy. The corresponding proteins are co-expressed in broad regions of the brain and associate to heteromeric K(+) channels. These channels mediate M-type currents that regulate neuronal excitability. We investigated the basis for the increase in currents seen after co-expressing these subunits in Xenopus oocytes. Noise analysis and single channel recordings revealed a conductance of approximately 18 pS for KCNQ2 and approximately 7 pS for KCNQ3. Different conductance levels (ranging from 8 to 22 pS) were seen upon co-expression. Their weighted average is close to that obtained by noise analysis (16 pS). The open probability of heteromeric channels was not increased significantly. Co-expression of both subunits increased the surface expression of KCNQ2 and KCNQ3 by factors of 5 and >10, respectively. A KCNQ2 mutant associated with BFNC that has a truncated cytoplasmic carboxyl terminus did not reach the surface and failed to stimulate KCNQ3 surface expression. By contrast, several BFNC-associated missense mutations in KCNQ2 or KCNQ3 did not alter their surface expression. Thus, the increase in currents seen upon co-expressing KCNQ2 and KCNQ3 is predominantly due to an increase in surface expression, which is dependent on an intact carboxyl terminus.     

Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

KCNQ2/KCNQ3 channels are the molecular correlates of the neuronal M-channels, which play a major role in the control of neuronal excitability. Notably, they differ from homomeric KCNQ2 channels in their distribution pattern within neurons, with unique expression of KCNQ2 in axons and nerve terminals. Here, combined reciprocal coimmunoprecipitation and two-electrode voltage clamp analyses in Xenopus oocytes revealed a strong association of syntaxin 1A, a major component of the exocytotic SNARE complex, with KCNQ2 homomeric channels resulting in a ∼2-fold reduction in macroscopic conductance and ∼2-fold slower activation kinetics. Remarkably, the interaction of KCNQ2/Q3 heteromeric channels with syntaxin 1A was significantly weaker and KCNQ3 homomeric channels were practically resistant to syntaxin 1A. Analysis of different KCNQ2 and KCNQ3 chimeras and deletion mutants combined with in-vitro binding analysis pinpointed a crucial C-terminal syntaxin 1A-association domain in KCNQ2. Pull-down and coimmunoprecipitation analyses in hippocampal and cortical synaptosomes demonstrated a physical interaction of brain KCNQ2 with syntaxin 1A, and confocal immunofluorescence microscopy showed high colocalization of KCNQ2 and syntaxin 1A at presynaptic varicosities. The selective interaction of syntaxin 1A with KCNQ2, combined with a numerical simulation of syntaxin 1A''s impact in a firing-neuron model, suggest that syntaxin 1A''s interaction is targeted at regulating KCNQ2 channels to fine-tune presynaptic transmitter release, without interfering with the function of KCNQ2/3 channels in neuronal firing frequency adaptation.     

Channel-anchored Protein Kinase CK2 and Protein Phosphatase 1 Reciprocally Regulate KCNQ2-containing M-channels via Phosphorylation of Calmodulin

M-type potassium channels, encoded by the KCNQ family genes (KCNQ2–5), require calmodulin as an essential co-factor. Calmodulin bound to the KCNQ2 subunit regulates channel trafficking and stabilizes channel activity. We demonstrate that phosphorylation of calmodulin by protein kinase CK2 (casein kinase 2) rapidly and reversibly modulated KCNQ2 current. CK2-mediated phosphorylation of calmodulin strengthened its binding to KCNQ2 channel, caused resistance to phosphatidylinositol 4,5-bisphosphate depletion, and increased KCNQ2 current amplitude. Accordingly, application of CK2-selective inhibitors suppressed KCNQ2 current. This suppression was prevented by co-expression of CK2 phosphomimetic calmodulin mutants or pretreatment with a protein phosphatase inhibitor, calyculin A. We also demonstrated that functional CK2 and protein phosphatase 1 (PP1) were selectively tethered to the KCNQ2 subunit. We identified a functional KVXF consensus site for PP1 binding in the N-terminal tail of KCNQ2 subunit: mutation of this site augmented current density. CK2 inhibitor treatment suppressed M-current in rat superior cervical ganglion neurons, an effect negated by overexpression of phosphomimetic calmodulin or pretreatment with calyculin A Furthermore, CK2 inhibition diminished the medium after hyperpolarization by suppressing the M-current. These findings suggest that CK2-mediated phosphorylation of calmodulin regulates the M-current, which is tonically regulated by CK2 and PP1 anchored to the KCNQ2 channel complex.     

The identification and characterization of a noncontinuous calmodulin-binding site in noninactivating voltage-dependent KCNQ potassium channels

We show here that in a yeast two-hybrid assay calmodulin (CaM) interacts with the intracellular C-terminal region of several members of the KCNQ family of potassium channels. CaM co-immunoprecipitates with KCNQ2, KCNQ3, or KCNQ5 subunits better in the absence than in the presence of Ca2+. Moreover, in two-hybrid assays where it is possible to detect interactions with apo-CaM but not with Ca2+-bound calmodulin, we localized the CaM-binding site to a region that is predicted to contain two alpha-helices (A and B). These two helices encompass approximately 85 amino acids, and in KCNQ2 they are separated by a dispensable stretch of approximately 130 amino acids. Within this CaM-binding domain, we found an IQ-like CaM-binding motif in helix A and two overlapping consensus 1-5-10 CaM-binding motifs in helix B. Point mutations in helix A or B were capable of abolishing CaM binding in the two-hybrid assay. Moreover, glutathione S-transferase fusion proteins containing helices A and B were capable of binding to CaM, indicating that the interaction with KCNQ channels is direct. Full-length CaM (both N and C lobes) and a functional EF-1 hand were required for these interactions to occur. These observations suggest that apo-CaM is bound to neuronal KCNQ channels at low resting Ca2+ levels and that this interaction is disturbed when the [Ca2+] is raised. Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of KCNQ channels.     

Regulation of the voltage-gated K(+) channels KCNQ2/3 and KCNQ3/5 by serum- and glucocorticoid-regulated kinase-1

The voltage-gated KCNQ2/3 and KCNQ3/5 K(+) channels regulate neuronal excitability. We recently showed that KCNQ2/3 and KCNQ3/5 channels are regulated by the ubiquitin ligase Nedd4-2. Serum- and glucocorticoid-regulated kinase-1 (SGK-1) plays an important role in regulation of epithelial ion transport. SGK-1 phosphorylation of Nedd4-2 decreases the ability of Nedd4-2 to ubiquitinate the epithelial Na(+) channel, which increases the abundance of channel protein in the cell membrane. In this study, we investigated the mechanism(s) of SGK-1 regulation of M-type KCNQ channels expressed in Xenopus oocytes. SGK-1 significantly upregulated the K(+) current amplitudes of KCNQ2/3 and KCNQ3/5 channels approximately 1.4- and approximately 1.7-fold, respectively, whereas the kinase-inactive SGK-1 mutant had no effect. The cell surface levels of KCNQ2-hemagglutinin/3 were also increased by SGK-1. Deletion of the KCNQ3 channel COOH terminus in the presence of SGK-1 did not affect the K(+) current amplitude of KCNQ2/3/5-mediated currents. Coexpression of Nedd4-2 and SGK-1 with KCNQ2/3 or KCNQ3/5 channels did not significantly alter K(+) current amplitudes. Only the Nedd4-2 mutant (S448A)Nedd4-2 exhibited a significant downregulation of the KCNQ2/3/5 K(+) current amplitudes. Taken together, these results demonstrate a potential mechanism for regulation of KCNQ2/3 and KCNQ3/5 channels by SGK-1 regulation of the activity of the ubiquitin ligase Nedd4-2.     

PIP(2) activates KCNQ channels,and its hydrolysis underlies receptor-mediated inhibition of M currents

KCNQ channels belong to a family of potassium ion channels with crucial roles in physiology and disease. Heteromers of KCNQ2/3 subunits constitute the neuronal M channels. Inhibition of M currents, by pathways that stimulate phospholipase C activity, controls excitability throughout the nervous system. Here we show that a common feature of all KCNQ channels is their activation by the signaling membrane phospholipid phosphatidylinositol-bis-phosphate (PIP(2)). We show that wortmannin, at concentrations that prevent recovery from receptor-mediated inhibition of M currents, blocks PIP(2) replenishment to the cell surface. Moreover, we identify a C-terminal histidine residue, immediately proximal to the plasma membrane, mutation of which renders M channels less sensitive to PIP(2) and more sensitive to receptor-mediated inhibition. Finally, native or recombinant channels inhibited by muscarinic agonists can be activated by PIP(2). Our data strongly suggest that PIP(2) acts as a membrane-diffusible second messenger to regulate directly the activity of KCNQ currents.     

Calmodulin Activation Limits the Rate of KCNQ2 K+ Channel Exit from the Endoplasmic Reticulum

The potential regulation of protein trafficking by calmodulin (CaM) is a novel concept that remains to be substantiated. We proposed that KCNQ2 K⁺ channel trafficking is regulated by CaM binding to the C-terminal A and B helices. Here we show that the L339R mutation in helix A, which is linked to human benign neonatal convulsions, perturbs CaM binding to KCNQ2 channels and prevents their correct trafficking to the plasma membrane. We used glutathione S-transferase fused to helices A and B to examine the impact of this and other mutations in helix A (I340A, I340E, A343D, and R353G) on the interaction with CaM. The process appears to require at least two steps; the first involves the transient association of CaM with KCNQ2, and in the second, the complex adopts an “active” conformation that is more stable and is that which confers the capacity to exit the endoplasmic reticulum. Significantly, the mutations that we have analyzed mainly affect the stability of the active configuration of the complex, whereas Ca²⁺ alone appears to affect the initial binding step. The spectrum of responses from this collection of mutants revealed a strong correlation between adopting the active conformation and channel trafficking in mammalian cells. These data are entirely consistent with the concept that CaM bound to KCNQ2 acts as a Ca²⁺ sensor, conferring Ca²⁺ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function.M-type channels are generated by the KCNQ (Kv7) family of voltage-gated subtypes (1), and they are found throughout the nervous system where they fulfill dominant roles in the control of excitability and neural discharges (2). Like all Kv channels, the KCNQ α subunits share a common core structure of six transmembrane segments with a voltage sensing domain (S1–S4) and a pore domain (S5 and S6; see Fig. 1) (3). Sequence analysis predicts the presence of four helical regions (A–D) in all family members (4), and helices A and B constitute the binding site for calmodulin (CaM).⁸ CaM is a prototypical Ca²⁺ sensor that confers Ca²⁺ sensitivity to a wide array of proteins, including ion channels (5). CaM is thought to mediate Ca²⁺-dependent inhibition of KCNQ channels (6), and in addition, we have postulated that a direct association with CaM is required for KCNQ2 channels to exit the endoplasmic reticulum (7).Open in a separate windowFIGURE 1.Topological representation of a KCNQ subunit. The consensus IQ residues are shown in bold. Circles and squares correspond to the residues mutated here (the squares indicate the mutations causing BFNC). The boxes indicate the regions with a high probability of adopting an α helix configuration, and the thick lines delineate the region fused to GST.To gain a deeper understanding into the involvement of CaM in channel trafficking, we have studied this interaction in vitro with a set of CaM-binding site-specific mutants. Although we had previously explored the impact of some of these mutants on channel trafficking (7), here we have extended this study to the mutant L339R located in helix A. This mutation, as well as the R353G mutation in helix A, has been linked to benign familial neonatal convulsions (BFNC), a human epileptic syndrome of newborn children (2). Accordingly, we demonstrate that there is a strong correlation between the impact of these mutations on the adoption of an “active” conformation by CaM and channel subunit exit from the ER, lending further support to the concept that CaM is a critical regulator for the exit of the channel from the ER.     

KCNQ1/KCNE1 assembly,co-translation not required

Voltage-gated potassium channels are often assembled with accessory proteins which increases their functional diversity. KCNE proteins are small accessory proteins that modulate voltage-gated potassium (K_V) channels. Although the functional effects of various KCNE proteins have been described, many questions remain regarding their assembly with the pore-forming subunits. For example, while previous experiments with some K_V channels suggest that the association of the pore-subunit with the accessory subunits occurs co-translationally in the endoplasmic reticulum, it is not known whether KCNQ1 assembly with KCNE1 occurs in a similar manner to generate the medically important cardiac slow delayed rectifier current (IKs). In this study we used a novel approach to demonstrate that purified recombinant human KCNE1 protein (prKCNE1) modulates KCNQ1 channels heterologously expressed in Xenopus oocytes resulting in generation of IKs. Incubation of KCNQ1-expressing oocytes with cycloheximide did not prevent IKs expression following prKCNE1 injection. By contrast, incubation with brefeldin A prevented KCNQ1 modulation by prKCNE1. Moreover, injection of the trafficking-deficient KCNE1-L51H reduced KCNQ1 currents. Together, these observations indicate that while assembly of KCNE1 with KCNQ1 does not require co-translation, functional KCNQ1-prKCNE1 channels assemble early in the secretory pathway and reach the plasma membrane via vesicular trafficking.     

KCNQ5, a novel potassium channel broadly expressed in brain, mediates M-type currents

KCNQ2 and KCNQ3, both of which are mutated in a type of human neonatal epilepsy, form heteromeric potassium channels that are expressed in broad regions of the brain. The associated current may be identical to the M-current, an important regulator of neuronal excitability. We now show that the RNA encoding the novel KCNQ5 channel is also expressed in brain and in sympathetic ganglia where it overlaps largely with KCNQ2 and KCNQ3. In addition, it is expressed in skeletal muscle. KCNQ5 yields currents that activate slowly with depolarization and can form heteromeric channels with KCNQ3. Currents expressed from KCNQ5 have voltage dependences and inhibitor sensitivities in common with M-currents. They are also inhibited by M1 muscarinic receptor activation. A KCNQ5 splice variant found in skeletal muscle displays altered gating kinetics. This indicates a molecular diversity of channels yielding M-type currents and suggests a role for KCNQ5 in the regulation of neuronal excitability.     

Pivoting between Calmodulin Lobes Triggered by Calcium in the Kv7.2/Calmodulin Complex

Kv7.2 (KCNQ2) is the principal molecular component of the slow voltage gated M-channel, which strongly influences neuronal excitability. Calmodulin (CaM) binds to two intracellular C-terminal segments of Kv7.2 channels, helices A and B, and it is required for exit from the endoplasmic reticulum. However, the molecular mechanisms by which CaM controls channel trafficking are currently unknown. Here we used two complementary approaches to explore the molecular events underlying the association between CaM and Kv7.2 and their regulation by Ca²⁺. First, we performed a fluorometric assay using dansylated calmodulin (D-CaM) to characterize the interaction of its individual lobes to the Kv7.2 CaM binding site (Q2AB). Second, we explored the association of Q2AB with CaM by NMR spectroscopy, using ¹⁵N-labeled CaM as a reporter. The combined data highlight the interdependency of the N- and C-lobes of CaM in the interaction with Q2AB, suggesting that when CaM binds Ca²⁺ the binding interface pivots between the N-lobe whose interactions are dominated by helix B and the C-lobe where the predominant interaction is with helix A. In addition, Ca²⁺ makes CaM binding to Q2AB more difficult and, reciprocally, the channel weakens the association of CaM with Ca²⁺.     

Ionic permeation and conduction properties of neuronal KCNQ2/KCNQ3 potassium channels

Heteromeric KCNQ2/3 potassium channels are thought to underlie the M-current, a subthreshold potassium current involved in the regulation of neuronal excitability. KCNQ channel subunits are structurally unique, but it is unknown whether these structural differences result in unique conduction properties. Heterologously expressed KCNQ2/3 channels showed a permeation sequence of while showing a conduction sequence of A differential contribution of component subunits to the properties of heteromeric KCNQ2/3 channels was demonstrated by studying homomeric KCNQ2 and KCNQ3 channels, which displayed contrasting ionic selectivities. KCNQ2/3 channels did not exhibit an anomalous mole-fraction effect in mixtures of K(+) and Rb(+). However, extreme voltage-dependence of block by external Cs(+) was indicative of multi-ion pore behavior. Block of KCNQ2/3 channels by external Ba(2+) ions was voltage-independent, demonstrating unusual ionic occupation of the outer pore. Selectivity properties and block of KCNQ2 were altered by mutation of outer pore residues in a manner consistent with the presence of multiple ion-binding sites. KCNQ2/3 channel deactivation kinetics were slowed exclusively by Rb(+), whereas activation of KCNQ2/3 channels was altered by a variety of external permeant ions. These data indicate that KCNQ2/3 channels are multi-ion pores which exhibit distinctive mechanisms of ion conduction and gating.     

Roles of alternative splicing in the functional properties of inner ear-specific KCNQ4 channels

The function of the KCNQ4 channel in the auditory setting is crucial to hearing, underpinned by the finding that mutations of the channel result in an autosomal dominant form of nonsyndromic progressive high frequency hearing loss. The precise function of KCNQ4 in the inner ear has not been established. However, recently we demonstrated that there is differential expression among four splice variants of KCNQ4 (KCNQ4_v1-v4) along the tonotopic axis of the cochlea. Alternative splicing specifies the outcome of functional channels by modifying the amino acid sequences within the C terminus at a site designated as the membrane proximal region. We show that variations within the C terminus of splice variants produce profound differences in the voltage-dependent phenotype and functional expression of the channel. KCNQ4_v4 lacks exons 9-11, resulting in deletion of 54 amino acid residues adjacent to the S6 domain compared with KCNQ4_v1. Consequently, the voltage-dependent activation of KCNQ4_v4 is shifted leftward by approximately 20 mV, and the number of functional channels is increased severalfold compared with KCNQ4_v1. The properties of KCNQ4_v2 and KCNQ4_v3 fall between KCNQ4_v1 and KCNQ4_v4. Because of variations in the calmodulin binding domains of the splice variants, the channels are differentially modulated by calmodulin. Co-expression of these splice variants yielded current magnitudes suggesting that the channels are composed of heterotetramers. Indeed, a dominant negative mutant of KCNQ4_v1 cripples the currents of the entire KCNQ4 channel family. Furthermore, the dominant negative KCNQ4 mutant stifles the activity of KCNQ2-5, raising the possibility of a global disruption of KCNQ channel activity and the ensuing auditory phenotype.