KCNQ1 Subunits Research Articles

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.

Benign Familial Neonatal Convulsions Caused by Altered Gating of KCNQ2/KCNQ3 Potassium Channels

The muscarinic-regulated potassium current (M-current), formed by the heteromeric assembly of subunits encoded by the KCNQ2 and KCNQ3 genes, is a primary regulator of neuronal excitability; this regulation is accomplished by impeding repetitive firing and causing spike-frequency adaptation. Mutations in KCNQ2 or KCNQ3 cause benign familial neonatal convulsions (BFNC), a rare autosomal-dominant generalized epilepsy of newborns, by reducing the maximal current carried by the M-channels without affecting ion selectivity or gating properties. Here we show that KCNQ2/KCNQ3 channels carrying a novel BFNC-causing mutation leading to an arginine to tryptophan substitution in the voltage-sensing S4 domain of KCNQ2 subunits (R214W) displayed slower opening and faster closing kinetics and a decreased voltage sensitivity with no concomitant changes in maximal current or plasma membrane expression. These results suggest that mutation-induced gating alterations of the M-current may cause epilepsy in neonates.

Cause for excite-M-ent in adrenal chromaffin cells

Cause for excite-M-ent in adrenal chromaffin cells

M Channel KCNQ2 Subunits Are Localized to Key Sites for Control of Neuronal Network Oscillations and Synchronization in Mouse Brain

Mutations in the potassium channel subunit KCNQ2 lead to benign familial neonatal convulsions, a dominantly inherited form of generalized epilepsy. In heterologous cells, KCNQ2 expression yields voltage-gated potassium channels that activate slowly (tau, approximately 0.1 sec) at subthreshold membrane potentials. KCNQ2 associates with KCNQ3, a homolog, to form heteromeric channels responsible for the M current (I(M)) in superior cervical ganglion (SCG) neurons. Muscarinic acetylcholine and peptidergic receptors inhibit SCG I(M), causing slow EPSPs and enhancing excitability. Here, we use KCNQ2N antibodies, directed against a conserved N-terminal portion of the KCNQ2 polypeptide, to localize KCNQ2-containing channels throughout mouse brain. We show that KCNQ2N immunoreactivity, although widespread, is particularly concentrated at key sites for control of rhythmic neuronal activity and synchronization. In the basal ganglia, we find KCNQ2N immunoreactivity on somata of dopaminergic and parvalbumin (PV)-positive (presumed GABAergic) cells of the substantia nigra, cholinergic large aspiny neurons of the striatum, and GABAergic and cholinergic neurons of the globus pallidus. In the septum, GABAergic, purinergic, and cholinergic neurons that contribute to the septohippocampal and septohabenular pathways exhibit somatic KCNQ2 labeling. In the thalamus, GABAergic nucleus reticularis neurons that regulate thalamocortical oscillations show strong labeling. In the hippocampus, many PV-positive and additional PV-negative interneurons exhibit strong somatic staining, but labeling of pyramidal and dentate granule somata is weak. There is strong neuropil staining in many regions. In some instances, notably the hippocampal mossy fibers, evidence indicates this neuropil staining is presynaptic.

Divergent expression of delayed rectifier K(+) channel subunits during mouse heart development.

The repolarization phase of the cardiac action potential is dependent on transmembrane K(+) currents. The slow (I(Ks)) and fast (I(Kr)) components of the delayed-rectifier cardiac K(+) current are generated by pore-forming alpha subunits KCNQ1 and KCNH2, respectively, in association with regulatory beta-subunit KCNE1, KCNE2 and perphaps KCNE3. In the present study we have investigated the distribution of transcripts encoding these five potassium channel-forming subunits during mouse heart development as well as the protein distribution of KCNQ1 and KCNH2. KCNQ1 and KCNH2 mRNAs (and protein) are first expressed at embryonic day (E) 9.5, showing comparable levels of expression within the atrial and ventricular myocardium during the embryonic and fetal stages. In contrast, the beta-subunits display a more dynamic pattern of expression during development. KCNE1 expression is first observed at E9.5 throughout the entire myocardium and progressively is confined to the ventricular myocardium. With further development (E16.5), KCNE1 expression is mainly confined to the compact ventricular myocardium. KCNE2 is first expressed at E9.5 and it is restricted already to the atrial myocardium. KCNE3 is first expressed at E8.5 throughout the myocardium and with further development, it becomes restricted to the atrial myocardium. The fact that alpha subunits are homogeneously distributed within the myocardium, whereas the beta subunits display a regionalized expression profile during cardiac development, suggest that differences in the slow and fast component of the delayed-rectifier cardiac K(+) currents between the atrial and the ventricular cardiomyocytes are mainly determined by differential beta-subunit distribution.

Altered potassium balance and aldosterone secretion in a mouse model of human congenital long QT syndrome.

The voltage-dependent K(+) channel responsible for the slowly activating delayed K(+) current I(Ks) is composed of pore-forming KCNQ1 and regulatory KCNE1 subunits, which are mutated in familial forms of cardiac long QT syndrome. Because KCNQ1 and KCNE1 genes also are expressed in epithelial tissues, such as the kidneys and the intestine, we have investigated the adaptation of KCNE1-deficient mice to different K(+) and Na(+) intakes. On a normal K(+) diet, homozygous kcne1(-/-) mice exhibit signs of chronic volume depletion associated with fecal Na(+) and K(+) wasting and have lower plasma K(+) concentration and higher levels of aldosterone than wild-type mice. Although plasma aldosterone can be suppressed by low K(+) diets or stimulated by low Na(+) diets, a high K(+) diet provokes a tremendous increase of plasma aldosterone levels in kcne1(-/-) mice as compared with wild-type mice (7.1-fold vs. 1.8-fold) despite lower plasma K(+) in kcne1(-/-) mice. This exacerbated aldosterone production in kcne1(-/-) mice is accompanied by an abnormally high plasma renin concentration, which could partly explain the hyperaldosteronism. In addition, we found that KCNE1 and KCNQ1 mRNAs are expressed in the zona glomerulosa of adrenal glands where I(Ks) may directly participate in the control of aldosterone production by plasma K(+). These results, which show that KCNE1 and I(Ks) are involved in K(+) homeostasis, might have important implications for patients with I(Ks)-related long QT syndrome, because hypokalemia is a well known risk factor for the occurrence of torsades de pointes ventricular arrhythmia.

Properties of single M-type KCNQ2/KCNQ3 potassium channels expressed in mammalian cells.

1. The single channel properties of KCNQ2/KCNQ3 channels underlying neuronal voltage-dependent M-type potassium currents were studied in cell-attached patches from transfected Chinese hamster ovary (CHO) cells. Macroscopic currents produced by homo- and heteromeric KCNQ2/KCNQ3 channels were measured using the perforated-patch whole-cell technique. 2. Compared with heteromeric KCNQ2 + KCNQ3 channels, homomeric KCNQ2 channels had lower slope conductance (9.0 +/- 0.3 and 5.8 +/- 0.3 pS, respectively) and open probability at 0 mV (0.30 +/- 0.07 and 0.15 +/- 0.03, respectively), consistent with their 3.8-fold smaller macroscopic currents. By contrast, homomeric KCNQ3 channels had the same slope conductance (9.0 +/- 1.1 pS) as KCNQ2 + KCNQ3 channels, and higher open probability (0.59 +/- 0.11), inconsistent with their 12.7-fold smaller macroscopic currents. Thus, KCNQ2 and KCNQ3 subunits may play different roles in the expression of M-type currents, with KCNQ2 ensuring surface expression of underlying channels and KCNQ3 modifying their function. 3. Both in homo- and heteromeric KCNQ2/KCNQ3 channels the shut time distributions were fitted with three, and the open time distributions with two, exponential components. By measuring these and other parameters (e.g. conductance and open probability) KCNQ2/ KCNQ3 channels can be shown to resemble previously characterised neuronal M-type channels.

The cardiac K+ channel KCNQ1 is essential for gastric acid secretion

Gastric H+ secretion via the H+/K+-adenosine triphosphatase is coupled to the uptake of K+. However, the molecular identity of luminal K+ channels enabling K+ recycling in parietal cells is unknown. This study was aimed to investigate these luminal K+ channels. Acid secretion was measured in vivo and in vitro; KCNQ1 protein localization was assessed by immunofluorescence, and acid-sensitivity of KCNQ1 by patch-clamp. We identified KCNQ1, which is mutated in cardiac long QT syndrome, as a K+ channel located in tubulovesicles and apical membrane of parietal cells, where it colocalized with H+/K+-adenosine triphosphatase. Blockade of KCNQ1 current by 293B led to complete inhibition of acid secretion. The putative KCNQ1 subunits, KCNE2 and KCNE3, were abundant in human stomach; KCNE1, however, was absent. Coexpression of KCNE3/KCNQ1 in COS cells led to an acid-insensitive current; KCNE2/KCNQ1 was activated by low extracellular pH. We identified KCNQ1 as the missing luminal K+ channel in parietal cells and characterized its crucial role in acid secretion. Because KCNE3 and KCNE2 are expressed in human stomach, one or both are candidates to coassemble with KCNQ1 in parietal cells. Thus, stomach- and subunit-specific inhibitors of KCNQ1 might offer new therapeutical perspectives for peptic ulcer disease.

Identification of Specific Pore Residues Mediating KCNQ1 Inactivation: A NOVEL MECHANISM FOR LONG QT SYNDROME

KCNQ1 inactivation bears electrophysiological characteristics different from classical N- and C-type inactivation in Shaker-like potassium channels. However, the molecular site of KCNQ1 inactivation has not yet been determined. KCNQ2 channels do not exert a fast inactivation in contrast to KCNQ1 channels. By expressing functional chimeras between KCNQ1 and KCNQ2 in Xenopus oocytes, we mapped the region of this inactivation to transmembrane domain S5 and the pore loop H5 and finally narrowed down the site to positions Gly(272) and Val(307) in KCNQ1. Exchanging these two amino acids individually with the analogous KCNQ2 residue abolished inactivation. Furthermore, a KCNQ1-like inactivation was introduced into KCNQ2 by mutagenesis in the corresponding region, confirming its relevance for the inactivation process. As KCNQ1 inactivation involves the regions S5 and H5, it exhibits a geography distinct from N- or C-type inactivation. Native cardiac I(Ks) channels comprising KCNQ1 and accessory MinK subunits do not inactivate because of the functional interaction of KCNQ1 with MinK. Mutations in KCNQ1 can lead to long QT1 syndrome, an inherited form of arrhythmia. The long QT1 mutant KCNQ1(L273F) displays a pronounced KCNQ1 inactivation. Here we show that when expressing mutant I(Ks) channels formed from KCNQ1(L273F) and MinK, MinK association no longer eliminates KCNQ1 inactivation. This results in smaller repolarizing currents in the heart and therefore represents a novel mechanism leading to long QT syndrome.

KCNQ1/KCNE1 potassium channels in mammalian vestibular dark cells

The high [K +] in the inner ear endolymph is essential for mechanosensory transduction in hearing and balance. Several ion channels, including a slowly activating, voltage-dependent, outwardly conducting K + channel composed of the KCNQ1 (KvLQT1) and KCNE1 (IsK/minK) subunits, are expressed at the apical surface of vestibular dark cells. We investigated the underlying molecular mechanisms of this conductance using in situ hybridization, RT-PCR, and immunocytochemistry and by tracking the ultrastructural changes of vestibular structures in kcne1(−/−) mice. In the wild type mice, the KCNE1 and KCNQ1 proteins are expressed specifically at the apical membrane of dark cells, as early as gestational day (GD) 17 for KCNE1 while KCNQ1 mRNAs can be detected at GD 18. This is the first demonstration that the two protein components of this potassium channel co-localize in a polarized fashion at the cellular level. Although the vestibular end-organs are normal at birth in kcne1(−/−) mice, they begin to show modifications during postnatal development: we observed an increase in the height of the dark cells, in their number of mitochondria, and in basolateral membrane infoldings. Subsequently, the epithelium degenerates and the endolymphatic space collapses. Similar changes are known to occur in the cardio-auditory Jervell–Lange-Nielsen syndrome which is caused by mutations in the same channel.

A founder mutation of the potassium channel KCNQ1 in long QT syndrome: Implications for estimation of disease prevalence and molecular diagnostics

OBJECTIVESWe took advantage of the genetic isolate of Finns to characterize a common long QT syndrome (LQTS) mutation, and to estimate the prevalence of LQTS.BACKGROUNDThe LQTS is caused by mutations in different ion channel genes, which vary in their molecular nature from family to family.METHODSThe potassium channel gene KCNQ1 was sequenced in two unrelated Finnish patients with Jervell and Lange-Nielsen syndrome (JLNS), followed by genotyping of 114 LQTS probands and their available family members. The functional properties of the mutation were studied using a whole-cell patch-clamp technique.RESULTSWe identified a novel missense mutation (G589D or KCNQ1-Fin) in the C-terminus of the KCNQ1 subunit. The voltage threshold of activation for the KCNQ1-Fin channel was markedly increased compared to the wild-type channel. This mutation was present in homozygous form in two siblings with JLNS, and in heterozygous form in 34 of 114 probands with Romano-Ward syndrome (RWS) and 282 family members. The mean (± SD) rate-corrected QT intervals of the heterozygous subjects (n = 316) and noncarriers (n = 423) were 460 ± 40 ms and 410 ± 20 ms (p < 0.001), respectively.CONCLUSIONSA single missense mutation of the KCNQ1 gene accounts for 30% of Finnish cases with LQTS, and it may be associated with both the RWS and JLNS phenotypes of the syndrome. The relative enrichment of this mutation most likely represents a founder gene effect. These circumstances provide an excellent opportunity to examine how genetic and nongenetic factors modify the LQTS phenotype.

TEA(+)-sensitive KCNQ1 constructs reveal pore-independent access to KCNE1 in assembled I(Ks) channels.

I(Ks), a slowly activating delayed rectifier K(+) current through channels formed by the assembly of two subunits KCNQ1 (KvLQT1) and KCNE1 (minK), contributes to the control of the cardiac action potential duration. Coassembly of the two subunits is essential in producing the characteristic and physiologically critical kinetics of assembled channels, but it is not yet clear where or how these subunits interact. Previous investigations of external access to the KCNE1 protein in assembled I(Ks) channels relied on occlusion of the pore by extracellular application of TEA(+), despite the very low TEA(+) sensitivity (estimated EC(50) > 100 mM) of channels encoded by coassembly of wild-type KCNQ1 with the wild type (WT) or a series of cysteine-mutated KCNE1 constructs. We have engineered a high affinity TEA(+) binding site into the h-KCNQ1 channel by either a single (V319Y) or double (K318I, V319Y) mutation, and retested it for pore-delimited access to specific sites on coassembled KCNE1 subunits. Coexpression of either KCNQ1 construct with WT KCNE1 in Chinese hamster ovary cells does not alter the TEA(+) sensitivity of the homomeric channels (IC(50) approximately 0.4 mM [TEA(+)](out)), providing evidence that KCNE1 coassembly does not markedly alter the structure of the outer pore of the KCNQ1 channel. Coexpression of a cysteine-substituted KCNE1 (F54C) with V319Y significantly increases the sensitivity of channels to external Cd(2+), but neither the extent of nor the kinetics of the onset of (or the recovery from) Cd(2+) block was affected by [TEA(+)](o) at 10x the IC(50) for channel block. These data strongly suggest that access of Cd(2+) to the cysteine-mutated site on KCNE1 is independent of pore occlusion caused by TEA(+) binding to the outer region of the KCNE1/V319Y pore, and that KCNE1 does not reside within the pore region of the assembled channels.

KCNE2 confers background current characteristics to the cardiac KCNQ1 potassium channel

Mutations in HERG and KCNQ1 (or KVLQT1) genes cause the life-threatening Long QT syndrome. These genes encode K(+) channel pore-forming subunits that associate with ancillary subunits from the KCNE family to underlie the two components, I(Kr) and I(Ks), of the human cardiac delayed rectifier current I(K). The KCNE family comprises at least three members. KCNE1 (IsK or MinK) recapitulates I(Ks) when associated with KCNQ1, whereas it augments the amplitude of an I(Kr)-like current when co-expressed with HERG. KCNE3 markedly changes KCNQ1 as well as HERG current properties. So far, KCNE2 (MirP1) has only been shown to modulate HERG current. Here we demonstrate the interaction of KCNE2 with the KCNQ1 subunit, which results in a drastic change of KCNQ1 current amplitude and gating properties. Furthermore, KCNE2 mutations also reveal their specific functional consequences on KCNQ1 currents. KCNQ1 and HERG appear to share unique interactions with KCNE1, 2 and 3 subunits. With the exception of KCNE3, mutations in all these partner subunits have been found to lead to an increased propensity for cardiac arrhythmias.

Contribution of the IsK (MinK) Potassium Channel Subunit to Regulatory Volume Decrease in Murine Tracheal Epithelial Cells

The cell volume regulatory response following hypotonic shocks is often achieved by the coordinated activation of K(+) and Cl(-) channels. In this study, we investigate the identity of the K(+) and Cl(-) channels that mediate the regulatory volume decrease (RVD) in ciliated epithelial cells from murine trachea. RVD was inhibited by tamoxifen and 1,9-dideoxyforskolin, two agents that block swelling-activated Cl(-) channels. These data suggest that swelling-activated Cl(-) channels play an important role in cell volume regulation in murine tracheal epithelial cells. Ba(2+) and apamin, inhibitors of K(+) channels, were without effect on RVD, while tetraethylammoniun had little effect on RVD. In contrast, clofilium, an inhibitor of the KvLQT/IsK potassium channel complex potently inhibited RVD, suggesting a role for the KvLQT/IsK channel complex in cell volume regulation by tracheal epithelial cells. To investigate further the role of KvLQT/IsK channels in RVD, we used IsK knock-out mice. When exposed to hypotonic solutions, tracheal cells from IsK(+/+) mice underwent RVD, whereas cells from IsK(-/-) failed to recover their normal size. These data suggest that the IsK potassium subunit plays an important role in RVD in murine tracheal epithelial cells.

Hypokalemia‐Induced Long QT Syndrome with an Underlying Novel Missense Mutation in S4‐S5 Linker of KCNQ1

Congenital long QT syndrome (LQTS) is caused by mutations in at least five genes coding for cardiac potassium or sodium channels that regulate the duration of ventricular action potentials. Acquired LQTS often is associated with drugs or metabolic abnormalities. A 47-year-old woman who presented with marked QT prolongation (QTc = 620 msec(1/2)) and repeated episodes of torsades de pointes associated with hypokalemia (2.6 mEq/L) was screened for mutations in LQTS genes using polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP). We identified a novel missense mutation in the intracellular linker of S4-S5 domains of KCNQ1, resulting in an amino acid substitution of cysteine for arginine at position 259 (R259C). Whole cell, patch clamp experiments were conducted on COS7 cells transfected with wild-type and/or R259C KCNQ1 with or without KCNE1. Functional analyses of the mutant KCNQ1 subunit on COS7 cells revealed its functional channels in the homozygous state, producing a significantly smaller current than the KCNQ1 channels and a less severe dominant-negative effect on I(Ks). The novel KCNQ1 mutation R259C is the molecular basis for I(Ks) dysfunction underlying an apparently sporadic case of hypokalemia-induced LQTS, consistent with a mild mutation likely to disclose the clinical manifestation of LQTS in a context of severe hypokalemia. Our findings suggest that gene carriers with such mild mutations might not be so rare as commonly expected in patients with acquired LQTS, and stress the importance of mutational analysis for detecting either "silent" forms of congenital LQTS or de novo mutations.

M-type KCNQ2–KCNQ3 potassium channels are modulated by the KCNE2 subunit

KCNQ2 and KCNQ3 subunits belong to the six transmembrane domain K + channel family and loss of function mutations are associated with benign familial neonatal convulsions. KCNE2 (MirP1) is a single transmembrane domain subunit first described to be a modulator of the HERG potassium channel in the heart. Here, we show that KCNE2 is present in brain, in areas which also express KCNQ2 and KCNQ3 channels. We demonstrate that KCNE2 associates with KCNQ2 and/or KCNQ3 subunits. In transiently transfected COS cells, KCNE2 expression produces an acceleration of deactivation kinetics of KCNQ2 and of the KCNQ2–KCNQ3 complex. Effects of two previously identified arrhythmogenic mutations of KCNE2 have also been analyzed.

Molecular Identity of the M-Channel

The M-current is a slowly activating and slowly deactivating potassium conductance that plays a critical role in determining the subthreshold electrical excitability of neurons (1-3). The M-current was first described in peripheral sympathetic neurons (4,5), where it is inhibited by stimulation of muscarinic acetylcholine receptors. Inhibition of the M-current and its subsequent effects on neuronal firing properties was one of the first clear descriptions of the mechanism by which neuromodulatory neurotransmitters act in the nervous system (4). Differential expression of the M-current produces subtypes of sympathetic neurons with distinct firing patterns (3). Sympathetic neurons that express the M-current have phasic firing properties, whereas neurons that lack the M-current have tonic firing properties. The M-current also is expressed in many neurons in the central nervous system (1). The molecular identity of the channels that underlie the M-current has recently been determined (6). In sympathetic neurons the M-current is formed by heteromultimers of the KCNQ2 and KCNQ3 potassium channel subunits. The KCNQ subfamily of potassium channel genes contains four members and is unusual because all four genes have been linked to human genetic diseases. The KC.NQ2 and KCNQ3 genes have both been linked to an inherited autosomal dominant epilepsy (7-9). The very similar phenotypes produced by mutation of either of these two distinct genes (10) can be explained by the observation that both gene products are required to produce full expression of functional M-channels (6). The KCNQ2+KCNQ3 channels have closely comparable properties to the native M-channel. The KCNQ2+KCNQ3 channel is the only known potassium channel that can reproduce the unique kinetic properties of the native M-current (6). The pharmacologic properties of the native M-current and the KCNQ2+KCNQ3 channel also are very similar. in particular, the compound XE991 has been shown to be a highly selective blocker of both the M-current and KCNQ channels (6). In addition, the KCNQ2 gene is the only known potassium channel gene expressed in a pattern that parallels the distribution of the M-current in peripheral sympathetic ganglia (6). The KCNQ2 and KCNQ3 genes also are abundantly expressed in the CNS, and it is likely that the KCNQ2+KCNQ3 subunits contribute to the M-current in many central neurons. The M-current, together with the calcium-activated potassium current known as the after-hyperpolarization current, are thought to be the two prirnnry determinants of neuronal firing patterns (2,3). The critical role of the M-current in controlling neuronal excitability is demonstrated in Fig. 1. After blockade of the M-current with XE991, a phasic firing sympathetic neuron is converted to a tonic firing pattern. The cell is considerably more excitable due to the inhibition of the subthreshold M-current. The strong effect of the M-current on neuronal excitability is consistent with the linkage of the KCNQ2 and KCNQ3 genes to an inherited epilepsy (7-9). Identification of the physiologic function of the channel encoded by the KCNQ2 and KCNQ3 genes may facilitate the development of symptomatic treatments for several epilepsies. In particular, compounds that act as enhancers of M-current function may have potential as antiepileptic drugs by dampening intrinsic electrical excitability.

Reconstitution of Muscarinic Modulation of the KCNQ2/KCNQ3 K+Channels That Underlie the Neuronal M Current

Channels from KCNQ2 and KCNQ3 genes have been suggested to underlie the neuronal M-type K(+) current. The M current is modulated by muscarinic agonists via G-proteins and an unidentified diffusible cytoplasmic messenger. Using whole-cell clamp, we studied tsA-201 cells in which cloned KCNQ2/KCNQ3 channels were coexpressed with M(1) muscarinic receptors. Heteromeric KCNQ2/KCNQ3 currents were modulated by the muscarinic agonist oxotremorine-M (oxo-M) in a manner having all of the characteristics of modulation of native M current in sympathetic neurons. Oxo-M also produced obvious intracellular Ca(2+) transients, observed by using indo-1 fluorescence. However, modulation of the current remained strong even when Ca(2+) signals were abolished by the combined use of strong intracellular Ca(2+) buffers, an inhibitor of IP(3) receptors, and thapsigargin to deplete Ca(2+) stores. Muscarinic modulation was not blocked by staurosporine, a broad-spectrum protein kinase inhibitor, arguing against involvement of protein kinases. The modulation was not associated with a shift in the voltage dependence of channel activation. Homomeric KCNQ2 and KCNQ3 channels also expressed well and were modulated individually by oxo-M, suggesting that the motifs for modulation are present on both channel subtypes. Homomeric KCNQ2 and KCNQ3 currents were blocked, respectively, at very low and at high concentrations of tetraethylammonium ion. Finally, when KCNQ2 subunits were overexpressed by intranuclear DNA injection in sympathetic neurons, total M current was fully modulated by the endogenous neuronal muscarinic signaling mechanism. Our data further rule out Ca(2+) as the diffusible messenger. The reconstitution of muscarinic modulation of the M current that uses cloned components should facilitate the elucidation of the muscarinic signaling mechanism.

Inhibition of KCNQ1-4 potassium channels expressed in mammalian cells via M1 muscarinic acetylcholine receptors.

1. KCNQ1-4 potassium channels were expressed in mammalian Chinese hamster ovary (CHO) cells stably transfected with M1 muscarinic acetylcholine receptors and currents were recorded using the whole-cell perforated patch technique and cell-attached patch recording. 2. Stimulation of M1 receptors by 10 microM oxotremorine-M (Oxo-M) strongly reduced (to 0-10%) currents produced by KCNQ1-4 subunits expressed individually and also those produced by KCNQ2 + KCNQ3 and KCNQ1 + KCNE1 heteromers, which are thought to generate neuronal M-currents (IK,M) and cardiac slow delayed rectifier currents (IK,s), respectively. 3. The activity of KCNQ2 + KCNQ3, KCNQ2 and KCNQ3 channels recorded with cell-attached pipettes was strongly and reversibly reduced by Oxo-M applied to the extra-patch membrane. 4. It is concluded that M1 receptors couple to all known KCNQ subunits and that inhibition of KCNQ2 + KCNQ3 channels, like that of native M-channels, requires a diffusible second messenger.

Differential tetraethylammonium sensitivity of KCNQ1–4 potassium channels

In Shaker-group potassium channels the presence of a tyrosine residue, just downstream of the pore signature sequence GYG, determines sensitivity to tetraethylammonium (TEA). The KCNQ family of channels has a variety of amino acid residues in the equivalent position. We studied the effect of TEA on currents generated by KCNQ homomers and heteromers expressed in CHO cells. We used wild-type KCNQ1-4 channels and heteromeric KCNQ2/3 channels incorporating either wild-type KCNQ3 subunits or a mutated KCNQ3 in which tyrosine replaced threonine at position 323 (mutant T323Y). IC50 values were (mM): KCNQ1, 5.0; KCNQ2, 0.3; KCNQ3, > 30; KCNQ4, 3.0; KCNQ2 + KCNQ3, 3.8; and KCNQ2 + KCNQ3(T323Y), 0.5. While the high TEA sensitivity of KCNQ2 may be conferred by a tyrosine residue lacking in the other channels, the intermediate TEA sensitivity of KCNQ1 and KCNQ4 implies that other residues are also important in determining TEA block of the KCNQ channels.