Mutations in CLCN2 encoding a voltage-gated chloride channel are associated with idiopathic generalized epilepsies.

Idiopathic generalized epilepsy (IGE) is an inherited neurological disorder affecting about 0.4% of the world's population. Mutations in ten genes causing distinct forms of idiopathic epilepsy have been identified so far, but the genetic basis of many IGE subtypes is still unknown. Here we report a gene associated with the four most common IGE subtypes: childhood and juvenile absence epilepsy (CAE and JAE), juvenile myoclonic epilepsy (JME), and epilepsy with grand mal seizures on awakening (EGMA; ref. 8). We identified three different heterozygous mutations in the chloride-channel gene CLCN2 in three unrelated families with IGE. These mutations result in (i) a premature stop codon (M200fsX231), (ii) an atypical splicing (del74-117) and (iii) a single amino-acid substitution (G715E). All mutations produce functional alterations that provide distinct explanations for their pathogenic phenotypes. M200fsX231 and del74-117 cause a loss of function of ClC-2 channels and are expected to lower the transmembrane chloride gradient essential for GABAergic inhibition. G715E alters voltage-dependent gating, which may cause membrane depolarization and hyperexcitability.

Endogenous ADP-ribose enables calcium-regulated cation currents through TRPM2 channels in neutrophil granulocytes.

TRPM2 (transient receptor potential melastatin 2) is a Ca2+-permeable cation channel gated by ADPR (ADP-ribose) from the cytosolic side. To test whether endogenous concentrations of intracellular ADPR are sufficient for TRPM2 gating in neutrophil granulocytes, we devised an HPLC method to determine ADPR contents in HClO4 cell extracts. The reversed-phase ion-pair HPLC method with an Mg2+-containing isocratic eluent allows baseline resolution of one ADPR peak. Intracellular ADPR concentrations were approx. 5 muM in granulocytes and not significantly altered by stimulation with the chemoattractant peptide fMLP (N-formylmethionyl-leucylphenylalanine). We furthermore determined intracellular concentrations of cADPR (cyclic ADPR) with a cyclase assay involving enzymatic conversion of cADPR into NAD+ and fluorimetric determination of NAD+. Intracellular cADPR concentrations were approx. 0.2 microM and not altered by fMLP. In patch-clamp experiments, ADPR (0.1-100 microM) was dialysed into granulocytes to analyse its effects on whole-cell currents characteristic for TRPM2, in the presence of a low (<10 nM) or a high (1 microM) intracellular Ca2+ concentration. TRPM2 currents were significantly larger at high than at low [Ca2+] (e.g. -225+/-27.1 versus -7+/-2.0 pA/pF at 5 muM ADPR), but no currents at all were observed in the absence of ADPR (ADPR concentration < or =0.3 microM). cADPR (0.1, 0.3 and 10 microM) was without effect even in the presence of subthreshold ADPR (0.1 microM). We conclude that ADPR enables an effective regulation of TRPM2 by cytosolic Ca2+. Thus ADPR and Ca2+ in concert behave as a messenger system for agonist-induced influx of Ca2+ through TRPM2 in granulocytes.

The myotonia congenita mutation A331T confers a novel hyperpolarization-activated gate to the muscle chloride channel ClC-1.

Mutations in the muscle chloride channel gene CLCN1 cause myotonia congenita, an inherited disorder of skeletal muscle excitability leading to a delayed relaxation after muscle contraction. Here, we examine the functional consequences of a novel disease-causing mutation that predicts the substitution of alanine by threonine at position 331 (A331T) by whole-cell patch-clamp recording of recombinant mutant channels. A331T hClC-1 channels exhibit a novel slow gate that activates during membrane hyperpolarization and closes at positive potentials. This novel gate acts in series with fast opening and closing transitions that are common to wild-type (WT) and mutant channels. Under conditions at which this novel gate is not activated, i.e., a holding potential of 0 mV, the typical depolarization-induced activation gating of WT hClC-1 was only slightly affected by the mutation. In contrast, A331T hClC-1 channels with an open slow gate display an altered voltage dependence of open probability. These novel gating features of mutant channels produce a decreased open probability at -85 mV, the normal muscle resting potential, leading to a reduced resting chloride conductance of affected muscle fibers. The A331T mutation causes an unprecedented alteration of ClC-1 gating and reveals novel processes defining transitions between open and closed states in ClC chloride channels.

Functional characterization of Ih-channel splice variants from Apis mellifera.

We isolated splice variants of the AMIH cDNA by means of polymerase chain reaction and homology screening. Splicing at one site generates at least four different channel transcripts (AMIH, AMIHL, AMIHM and AMIHT), which code for ion-channel proteins that vary in the interloop regions between the membrane-spanning domains S4 and S5. HEK293 cells in which the AMIHL splice variants were functionally expressed generated currents that were activated by hyperpolarizing voltage steps. Compared to AMIH, AMIHL cells showed pronounced differences in the voltage dependency of activation: the incorporation of 32 extra amino acids between S4 and S5 shifts the activation curve by +25 mV. Intracellular cAMP made the current-activation potential still less negative and accelerated the activation more effectively than it does in AMIH cells. In vertebrates, functional diversity of Ih-channels is generated by four different genes. In Apis mellifera, splice variants coded by the single gene AMIH could generate a similar diversity.

Characterization of recombinant and native Ih-channels from Apis mellifera.

Recently, a novel class of genes coding for Ih-channels has been identified in several vertebrates and invertebrates. We isolated a cDNA (AMIH) encoding a putative member of these ion channels from Apis mellifera heads by means of polymerase chain reaction and homology screening. High similarity (88% identical amino acids) to the putative Drosophila melanogaster Ih-channel suggests that the Apis cDNA codes for a hyperpolarization-activated and cyclic nucleotide-gated channel. Functional expression of recombinant AMIH in HEK293 cells gave unitary currents that were preferentially selective for potassium over sodium ions and were activated by hyperpolarizing voltage steps. Cyclic nucleotides shifted the voltage activation curve to more positive membrane potentials. The current kinetics, activation by hyperpolarizing voltage steps and modulatory influence of cyclic nucleotides properties closely resemble those of mammalian Ih-channels. RT-PCR analysis showed pronounced mRNA expression in the antennae, head and body of Apis mellifera. Investigation of hyperpolarization-activated currents in olfactory receptor neurons (ORNs) in a primary cell culture of Apis mellifera antennal cells revealed activation properties similar to the heterologously expressed Ih-channel. By in-situ hybridization and immunohistochemistry, expression of AMIH was seen in olfactory receptor neurons of the bee antennae. We conclude that AMIH is the ion channel responsible for the hyperpolarization-activated currents in olfactory receptor neurons of bee.

Novel CLCN1 mutations with unique clinical and electrophysiological consequences.

Myotonia is a condition characterized by impaired relaxation of muscle following sudden forceful contraction. We systematically screened all 23 exons of the CLCN1 gene in 88 unrelated patients with myotonia and identified mutations in 14 patients. Six novel mutations were discovered: five were missense (S132C, L283F, T310M, F428S and T550M) found in heterozygous patients, and one was a nonsense mutation (E193X) in a homozygous patient. While five patients had a clinical diagnosis of myotonia congenita, the patient with the F428S mutation exhibited symptoms characteristic of paramyotonia congenita--a condition usually thought to be caused by mutations in the sodium channel gene SCN4A. Nevertheless, no mutations in SCN4A were identified in this patient. The functional consequences of the novel CLCN1 sequence variants were explored by recording chloride currents from human embryonic kidney cells transiently expressing homo- or heterodimeric mutant channels. The five tested mutations caused distinct functional alterations of the homodimeric human muscle chloride ion channel hClC-1. S132C and T550M conferred novel hyperpolarization-induced gating steps, L283F and T310M caused a shift of the activation curve to more positive potentials and F428S reduced the expression level of hClC-1 channels. All showed a dominant-negative effect. For S132C, L283F, T310M and T550M, heterodimeric channels consisting of one wild-type (WT) and one mutant subunit exhibited a shifted activation curve at low intracellular [Cl(-)]. WT-F428S channels displayed properties similar to WT hClC-1, but expressed at significantly lower levels. The novel mutations exhibit a broad variety of functional defects that, by distinct mechanisms, cause a significant reduction of the resting chloride conductance in muscle of heterozygous patients. Our results provide novel insights into functional alterations and clinical symptoms caused by mutations in CLCN1.