Bernard Attali

A recessive C-terminal Jervell and Lange-Nielsen mutation of the KCNQ1 channel impairs subunit assembly

The LQT1 locus (KCNQ1) has been correlated with the most common form of inherited long QT (LQT) syndrome. LQT patients suffer from syncopal episodes and high risk of sudden death. The KCNQ1 gene encodes KvLQT1 α‐subunits, which together with auxiliary IsK (KCNE1, minK) subunits form IKs K+ channels. Mutant KvLQT1 subunits may be associated either with an autosomal dominant form of inherited LQT, Romano–Ward syndrome, or an autosomal recessive form, Jervell and Lange‐Nielsen syndrome (JLNS). We have identified a small domain between residues 589 and 620 in the KvLQT1 C‐terminus, which may function as an assembly domain for KvLQT1 subunits. KvLQT1 C‐termini do not assemble and KvLQT1 subunits do not express functional K+ channels without this domain. We showed that a JLN deletion–insertion mutation at KvLQT1 residue 544 eliminates important parts of the C‐terminal assembly domain. Therefore, JLN mutants may be defective in KvLQT1 subunit assembly. The results provide a molecular basis for the clinical observation that heterozygous JLN carriers show slight cardiac dysfunctions and that the severe JLNS phenotype is characterized by the absence of KvLQT1 channel.

Cloning, expression, pharmacology and regulation of a delayed rectifier K+ channel in mouse heart.

Neonatal mouse cardiac poly(A)+ mRNA microinjection into Xenopus oocytes directed the expression of a delayed rectifier K+ current. A cDNA encoding this channel, called mIsK, was cloned from a neonatal mouse heart cDNA library whose properties were studied after expression of the complementary RNA in Xenopus oocytes. Among the different known K+ channel blockers, only the class III antiarrhythmic clofilium inhibited mIsK in the 10–100 microM range. The channel was completely insensitive to other antiarrhythmics such as quinine, quinidine, sotalol or amiodarone. mIsK was enhanced by increasing intracellular Ca2+ and by microinjected Ca(2+)‐calmodulin dependent protein kinase II. These stimulations were reversed by the calmodulin antagonist W7. Conversely, the phorbol ester PMA, the diacylglycerol analog OAG and microinjected purified protein kinase C inhibited mIsK. This inhibitory effect could be prevented by the protein kinase C inhibitor staurosporine. These results were consistent with the presence of consensus sequences for kinase II and kinase C in the mIsK structure. Cultured newborn mouse ventricular cardiac cells exhibited a delayed rectifier K+ current which had biophysical properties similar to those of cloned mIsK and which was inhibited by clofilium and protein kinase C activators. In situ hybridization experiments revealed that mIsK mRNA was homogeneously distributed in the cardiac tissue. Neonatal mouse heart expressed the most mIsK mRNA compared with various other rat and mouse tissues. Since this K+ channel generates a current which appears to be involved in the control of both the action potential duration and the beating rate, these results suggest an important role for the mIsK channel in cardiac cell physiology and cardiac pathology.

Molecular Mechanism and Functional Significance of the MinK Control of the KvLQT1 Channel Activity

The very slowly activating delayed rectifier K+ channel IKs is essential for controlling the repolarization phase of cardiac action potentials and K+ homeostasis in the inner ear. The IKschannel is formed via the assembly of two transmembrane proteins, KvLQT1 and MinK. Mutations in KvLQT1 are associated with a long QT syndrome that causes syncope and sudden death and also with deafness. Here, we show a new mode of association between ion channel forming subunits in that the cytoplasmic C-terminal end of MinK interacts directly with the pore region of KvLQT1. This interaction reduces KvLQT1 channel conductance from 7.6 to 0.58 picosiemens. However, because MinK also reveals a large number of previously silent KvLQT1 channels (× 60), the overall effect is a large increase (× 4) in the macroscopic K+ current. Conformational changes associated with the KvLQT1/MinK association create very slow and complex activation kinetics without much alteration in the deactivation process. Changes induced by MinK have an essential regulatory role in the development of this K+ channel activity upon repetitive electrical stimulation with a particular interest in tachycardia.

K‐Opiate Agonists Inhibit Adenylate Cyclase and Produce Heterologous Desensitization in Rat Spinal Cord

Abstract: The nature of the opiate modulation of adenylate cyclase following acute and chronic agonist exposure has been investigated in rat spinal cord. Using membranes of both adult rat spinal cord and spinal cord‐dorsal root ganglion cocultures, we found that K‐opiate receptors are negatively coupled to adenylate cyclase. The K‐opiate agonists (e.g., U50488) inhibit significantly and dose‐dependently the basal and the forskolin‐stimulated cyclase activities, whereas μ and δ agonists are ineffective. The regulatory action is stereospecific and requires the presence of GTP. EGTA treatment of the plasma membranes abolished the effect of K‐opiate agonists on the basal cyclase activity, and this inhibitory effect could not be restored by subsequent addition of Ca2+. The EGTA treatment did not affect the K agonist inhibition of the forolin‐stimulated cyclase. The results also show that following chronic exposure of cultured cells to etorphine or U50488, there is a loss of K agonist inhibition of the cyclase. Moreover, this desensitization process appears to be heterologous, because α2‐adrenergic agonists (e.g., clonidine or norepinephrine) and the muscarinic agonist (carbachol) exhibited significantly lower potency for inhibiting cyclase activity when compared to untreated cultures. This pattern of heterologous desensitization suggests that chronic exposure to K opiates leads to alterations in postreceptor regulatory components, possibly GTP‐binding proteins.

Hypomyelination and increased activity of voltage‐gated K + channels in mice lacking protein tyrosine phosphatase ϵ

Protein tyrosine phosphatase epsilon (PTPϵ) is strongly expressed in the nervous system; however, little is known about its physiological role. We report that mice lacking PTPϵ exhibit hypomyelination of sciatic nerve axons at an early post‐natal age. This occurs together with increased activity of delayed‐ rectifier, voltage‐gated potassium (Kv) channels and with hyperphosphorylation of Kv1.5 and Kv2.1 Kv channel α‐subunits in sciatic nerve tissue and in primary Schwann cells. PTPϵ markedly reduces Kv1.5 or Kv2.1 current amplitudes in Xenopus oocytes. Kv2.1 associates with a substrate‐trapping mutant of PTPϵ, and PTPϵ profoundly reduces Src‐ or Fyn‐stimulated Kv2.1 currents and tyrosine phosphorylation in transfected HEK 293 cells. In all, PTPϵ antagonizes activation of Kv channels by tyrosine kinases in vivo, and affects Schwann cell function during a critical period of Schwann cell growth and myelination.

Constitutive activation of delayed‐rectifier potassium channels by a Src family tyrosine kinase in Schwann cells

In the nervous system, Src family tyrosine kinases are thought to be involved in cell growth, migration, differentiation, apoptosis, as well as in myelination and synaptic plasticity. Emerging evidence indicates that K+ channels are crucial targets of Src tyrosine kinases. However, most of the data accumulated so far refer to heterologous expression, and native K+‐channel substrates of Src or Fyn in neurons and glia remain to be elucidated. The present study shows that a Src family tyrosine kinase constitutively activates delayed‐rectifier K+ channels (IK) in mouse Schwann cells (SCs). IK currents are markedly downregulated upon exposure of cells to the tyrosine kinase inhibitors herbimycin A and genistein, while a potent upregulation of IK is observed when recombinant Fyn kinase is introduced through the patch pipette. The Kv1.5 and Kv2.1 K+‐channel α subunits are constitutively tyrosine phosphorylated and physically associate with Fyn both in cultured SCs and in the sciatic nerve in vivo. Kv2.1‐ channel subunits are found to interact with the Fyn SH2 domain. Inhibition of Schwann cell proliferation by herbimycin A and by K+‐channel blockers suggests that the functional linkage between Src tyrosine kinases and IK channels could be important for Schwann cell proliferation and the onset of myelination.

Stilbenes and fenamates rescue the loss of I KS channel function induced by an LQT5 mutation and other IsK mutants

Genetic and physiological studies have established a link between potassium channel dysfunction and a number of neurological and muscular disorders. Many ‘channelopathies’ are accounted for by a dominant‐lethal suppression of potassium channel function. In the cardiac IKS channel complex comprising the α and β subunits, KvLQT1 and IsK, respectively, several mutations lead to a dominant‐negative loss of channel function. These defects are responsible for a human cardiovascular disease called long QT (LQT) syndrome. Here we show that binding of IKS channel activators, such as stilbenes and fenamates, to an extracellular domain flanking the human IsK transmembrane segment, restores normal IKS channel gating in otherwise inactive IsK C‐terminal mutants, including the naturally occurring LQT5 mutant, D76N. Our data support a model in which allosteric interactions exist between the extracellular and intracellular boundaries of the IsK transmembrane segment as well as between domains of the α and β subunits. Disruption of this allosteric interplay impedes slow activation gating, decreases current amplitude and restores channel inactivation. Owing to allosteric interactions, stilbene and fenamate compounds can rescue the dominant‐negative suppression of IKS produced by IsK mutations and thus, may have important therapeutic relevance for LQT syndrome.

Heteromultimeric Delayed-Rectifier K+ Channels in Schwann Cells: Developmental Expression and Role in Cell Proliferation

Schwann cells (SCs) are responsible for myelination of nerve fibers in the peripheral nervous system. Voltage-dependent K+ currents, including inactivating A-type (KA), delayed-rectifier (KD), and inward-rectifier (KIR) K+ channels, constitute the main conductances found in SCs. Physiological studies have shown that KD channels may play an important role in SC proliferation and that they are downregulated in the soma as proliferation ceases and myelination proceeds. Recent studies have begun to address the molecular identity of K+ channels in SCs. Here, we show that a large repertoire of K+ channel α subunits of theShaker (Kv1.1, Kv1.2, Kv1.4, and Kv1.5),Shab (Kv2.1), and Shaw (Kv3.1b and Kv3.2) families is expressed in mouse SCs and sciatic nerve. We characterized heteromultimeric channel complexes that consist of either Kv1.5 and Kv1.2 or Kv1.5 and Kv1.4. In postnatal day 4 (P4) sciatic nerve, most of the Kv1.2 channel subunits are involved in heteromultimeric association with Kv1.5. Despite the presence of Kv1.1 and Kv1.2 α subunits, the K+ currents were unaffected by dendrotoxin I (DTX), suggesting that DTX-sensitive channel complexes do not account substantially for SC KDcurrents. SC proliferation was found to be potently blocked by quinidine or 4-aminopyridine but not by DTX. Consistent with previous physiological studies, our data show that there is a marked downregulation of all KD channel α subunits from P1–P4 to P40 in the sciatic nerve. Our results suggest that KD currents are accounted for by a complex combinatorial activity of distinct K+channel complexes and confirm that KDchannels are involved in SC proliferation.

Different types of K+ channel current are generated by different levels of a single mRNA.

A cloned human voltage‐sensitive K+ channel HLK3 which is present in T‐lymphocytes and in the brain was expressed in Xenopus oocytes and after permanent transfection of a human B‐lymphocyte cell line (IM9). Injections of low cRNA concentrations into Xenopus oocytes led to the expression of a transient K+ current, with saturating current‐voltage (I‐V) relationship, which was abolished by repetitive stimulations due to a slow recovery from inactivation. This transient K+ channel current was fully inhibited by 10 nM charybdotoxin. Injection of high concentrations of the same RNA led to a non‐inactivating K+ current, with linear I‐V curve, which did not undergo use‐dependent inactivation and was hardly sensitive to 10 nM charybdotoxin. Intermediate behaviour due to changing proportions of these two types of K+ channel expression were observed at intermediate RNA concentrations. Transient and non‐inactivating K+ currents were also observed by both whole‐cell and single channel patch‐clamp recording from HLK3 transfected IM9 cells. The main conductance of the channel in the two different modes (inactivating and charybdotoxin‐sensitive or non‐inactivating and charybdotoxin‐resistant) is the same (12–14 pS). Destruction of the cytoskeletal elements with cytochalasin D, colchicine or botulinum C2 toxin in oocyte experiments prevented expression of the sustained mode of the K+ channel. The results suggest that the sustained mode obtained at high RNA concentrations corresponds to channel clustering involving cytoskeletal elements. This differential functional expression of K+ channels associated with different levels of mRNA appears as a new important factor to explain the biophysical and pharmacological diversity of voltage‐sensitive K+ channels.

Pre- and postnatal development of opiate receptor subtypes in rat spinal cord.

We have studied the developmental expression of opiate binding sites in the rat spinal cord at various prenatal and postnatal stages. For each developmental stage, we have compared the expression pattern of kappa receptors with that of mu and delta receptor subtypes. Both mu and kappa receptors appear relatively early during spinal cord ontogeny (from the 15th prenatal day), while delta sites are expressed later at the postnatal period (starting at the 1st postnatal day). The number of kappa sites predominates throughout the development (55-80% of total opiate sites) with two peaks of binding activity: one at the 20th gestational day, and the other around the 7th postnatal day. mu sites represent 20-38% of the total opiate receptor population with one peak of binding activity appearing at the 1st postnatal day. The densities of mu and kappa receptors at the adult stage are lower by 40-50% than the peak values observed at the early postnatal periods. The relative amounts of delta sites remain low throughout the ontogeny (4-8% of the total opiate sites). The binding properties of neonatal (1 day after birth) kappa sites (ligand binding affinities, regulation of agonist binding by guanosine triphosphate and various cations) are similar to those displayed by kappa receptors in adult spinal cord.