Kv7 channels tune neuronal and cardiomyocyte excitability. In addition to the channel membrane domain, they also have a unique intracellular C-terminal (CT) domain, bound constitutively to calmodulin (CaM). This CT domain regulates gating and tetramerization. We investigated the structure of the membrane proximal CT module in complex with CaM by X-ray crystallography. The results show how the CaM intimately hugs a two-helical bundle, explaining many channelopathic mutations. Structure-based mutagenesis of this module in the context of concatemeric tetramer channels and functional analysis along with in vitro data lead us to propose that one CaM binds to one individual protomer, without crosslinking subunits and that this configuration is required for proper channel expression and function. Molecular modeling of the CT/CaM complex in conjunction with small-angle X-ray scattering suggests that the membrane proximal region, having a rigid lever arm, is a critical gating regulator.

The voltage-dependent M-type potassium current (M-current) plays a major role in controlling brain excitability by stabilizing the membrane potential and acting as a brake for neuronal firing. The KCNQ2/Q3 heteromeric channel complex was identified as the molecular correlate of the M-current. Furthermore, the KCNQ2 and KCNQ3 channel ␣ subunits are mutated in families with benign familial neonatal convulsions, a neonatal form of epilepsy. Enhancement of KCNQ2/Q3 potassium currents may provide an important target for antiepileptic drug development. Here, we show that meclofenamic acid (meclofenamate) and diclofenac, two related molecules previously used as anti-inflammatory drugs, act as novel KCNQ2/Q3 channel openers. Extracellular application of meclofenamate (EC 50 ϭ 25 M) and diclofenac (EC 50 ϭ 2.6 M) resulted in the activation of KCNQ2/Q3 K ϩ currents, heterologously expressed in Chinese hamster ovary cells. Both openers activated KCNQ2/Q3 channels by causing a hyperpolarizing shift of the voltage activation curve (Ϫ23 and Ϫ15 mV, respectively) and by markedly slowing the deactivation kinetics. The effects of the drugs were stronger on KCNQ2 than on KCNQ3 channel ␣ subunits. In contrast, they did not enhance KCNQ1 K ϩ currents. Both openers increased KCNQ2/Q3 current amplitude at physiologically relevant potentials and led to hyperpolarization of the resting membrane potential. In cultured cortical neurons, meclofenamate and diclofenac enhanced the M-current and reduced evoked and spontaneous action potentials, whereas in vivo diclofenac exhibited an anticonvulsant activity (ED 50 ϭ 43 mg/kg). These compounds potentially constitute novel drug templates for the treatment of neuronal hyperexcitability including epilepsy, migraine, or neuropathic pain.Voltage-dependent K ϩ (Kv) channels play a major role in brain excitability through the regulation of action potential generation and propagation, the tuning of neuronal firing patterns, or the modulation of neurotransmitter release. The M-type K ϩ channel generates a subthreshold, voltage-gated K ϩ current (M-current) that plays an important role in controlling neuronal excitability. Brown and Adams (1980) first identified the M-current in frog sympathetic neurons as a slowly activating, noninactivating, voltage-sensitive K ϩ current, which was inhibited by muscarinic acetylcholine receptor stimulation (Brown and Adams, 1980). M-currents were also characterized in hippocampal and cortical neurons (Brown, 1988;Marrion, 1997;Cooper and Jan, 2003). Modulation of the M-current has profound effects on brain excitability because this noninactivating K ϩ channel exhibits significant conductance in the voltage range of action potential initiation. The low-threshold gating and the slow activation and deactivation of the M-current act as a brake for repetitive firing and neuronal excitability (

Abstract-The slow I KS K ϩ channel plays a major role in repolarizing the cardiac action potential and consists of the assembly of KCNQ1 and KCNE1 subunits. Mutations in either KCNQ1 or KCNE1 genes produce the long-QT syndrome, a life-threatening ventricular arrhythmia. Here, we show that long-QT mutations located in the KCNQ1 C terminus impair calmodulin (CaM) binding, which affects both channel gating and assembly. The mutations produce a voltage-dependent macroscopic inactivation and dramatically alter channel assembly. KCNE1 forms a ternary complex with wild-type KCNQ1 and Ca 2ϩ -CaM that prevents inactivation, facilitates channel assembly, and mediates a Ca 2ϩ -sensitive increase of I KS-current, with a considerable Ca 2ϩ -dependent left-shift of the voltage-dependence of activation. Coexpression of KCNQ1 or I KS channels with a Ca 2ϩ -insensitive CaM mutant markedly suppresses the currents and produces a right shift in the voltage-dependence of channel activation. KCNE1 association to KCNQ1 long-QT mutants significantly improves mutant channel expression and prevents macroscopic inactivation. However, the marked right shift in channel activation and the subsequent decrease in current amplitude cannot restore normal levels of I KS channel activity. Our data indicate that in healthy individuals, CaM binding to KCNQ1 is essential for correct channel folding and assembly and for conferring Ca 2ϩ -sensitive I KS -current stimulation, which increases the cardiac repolarization reserve and hence prevents the risk of ventricular arrhythmias. Key Words: KCNQ Ⅲ potassium channels Ⅲ Kv7 Ⅲ calmodulin Ⅲ KCNE Ⅲ long QT K CNQ channels represent a family of voltage-gated K ϩ channels (Kv7) that plays a major role in brain and cardiac excitability. 1,2 Mutations of human KCNQ genes lead to severe cardiovascular and neurological disorders such as the cardiac long-QT syndrome (LQT) and neonatal epilepsy. Coassembly of KCNQ1 with KCNE1 ␤ subunits produces the I KS -current that is crucial for repolarization of the cardiac action potential. [3][4][5] The cytoplasmic KCNQ C-termini were shown to feature 4 ␣ helices. 6 We previously identified the last ␣ helix of the C terminus (helix D, aa.589 -620) as a region important for the tetrameric assembly of KCNQ1 ␣ subunits. 7 This region also binds Yotiao, an A-kinase-anchoring protein that targets PKA on the I KS channel complex. 8 The first 2␣ helices of KCNQ1-5 form a calmodulin-binding domain (CBD), including an IQ motif that mediates Ca 2ϩ -free calmodulin (apoCaM) binding. 6,9 Although KCNQ channels bind calmodulin (CaM), the role of CaM in channel function remains controversial. Recent studies found a role for CaM as a Ca 2ϩ -sensor of KCNQ2/4/5 channels, 10,11 whereas others suggested a role in channel assembly. 9 So far, no information has been available about the interaction of calmodulin with cardiac I KS channels and its pathophysiological impact to KCNQ1-related LQT channnelopathies. Here, we show that LQT mutations located near the IQ motif of KCNQ1 C terminus impair ...

Elevated extracellular K+ ([K+]o), in the absence of “classical” immunological stimulatory signals, was found to itself be a sufficient stimulus to activate T cell β1 integrin moieties, and to induce integrin-mediated adhesion and migration. Gating of T cell voltage-gated K+ channels (Kv1.3) appears to be the crucial “decision-making” step, through which various physiological factors, including elevated [K+]o levels, affect the T cell β1 integrin function: opening of the channel leads to function, whereas its blockage prevents it. In support of this notion, we found that the proadhesive effects of the chemokine macrophage-inflammatory protein 1β, the neuropeptide calcitonin gene–related peptide (CGRP), as well as elevated [K+]o levels, are blocked by specific Kv1.3 channel blockers, and that the unique physiological ability of substance P to inhibit T cell adhesion correlates with Kv1.3 inhibition. Interestingly, the Kv1.3 channels and the β1 integrins coimmunoprecipitate, suggesting that their physical association underlies their functional cooperation on the T cell surface. This study shows that T cells can be activated and driven to integrin function by a pathway that does not involve any of its specific receptors (i.e., by elevated [K+]o). In addition, our results suggest that undesired T cell integrin function in a series of pathological conditions can be arrested by molecules that block the Kv1.3 channels.