Voltage-gated calcium channels represent a heterogenous family of calcium-selective channels that can be distinguished by their molecular, electrophysiological, and pharmacological characteristics. We report here the molecular cloning and functional expression of three members of the low voltage-activated calcium channel family from rat brain (␣ 1G , ␣ 1H , and ␣ 1I ). Northern blot and reverse transcriptase-polymerase chain reaction analyses show ␣ 1G , ␣ 1H , and ␣ 1I to be expressed throughout the newborn and juvenile rat brain. In contrast, while ␣ 1G and ␣ 1H mRNA are expressed in all regions in adult rat brain, ␣ 1I mRNA expression is restricted to the striatum. Expression of ␣ 1G , ␣ 1H , and ␣ 1I subunits in HEK293 cells resulted in calcium currents with typical T-type channel characteristics: low voltage activation, negative steady-state inactivation, strongly voltage-dependent activation and inactivation, and slow deactivation. In addition, the direct electrophysiological comparison of ␣ 1G , ␣ 1H , and ␣ 1I under identical recording conditions also identified unique characteristics including activation and inactivation kinetics and permeability to divalent cations. Simulation of ␣ 1G , ␣ 1H , and ␣ 1I T-type channels in a thalamic neuron model cell produced unique firing patterns (burst versus tonic) typical of different brain nuclei and suggests that the three channel types make distinct contributions to neuronal physiology.

T-type calcium channels play critical roles in cellular excitability and have been implicated in the pathogenesis of a variety of neurological disorders including epilepsy. Although there have been reports that certain neuroleptics that primarily target D2 dopamine receptors and are used to treat psychoses may also interact with T-type Ca channels, there has been no systematic examination of this phenomenon. In the present paper we provide a detailed analysis of the effects of several widely used neuroleptic agents on a family of exogenously expressed neuronal T-type Ca channels (alpha1G, alpha1H, and alpha1I subtypes). Among the neuroleptics tested, the diphenylbutylpiperidines pimozide and penfluridol were the most potent T-type channel blockers with Kd values (approximately 30-50 nm and approximately 70-100 nm, respectively), in the range of their antagonism of the D2 dopamine receptor. In contrast, the butyrophenone haloperidol was approximately 12- to 20-fold less potent at blocking the various T-type Ca channels. The diphenyldiperazine flunarizine was also less potent compared with the diphenylbutylpiperadines and preferentially blocked alpha1G and alpha1I T-type channels compared with alpha1H. The various neuroleptics did not significantly affect T-type channel activation or kinetic properties, although they shifted steady-state inactivation profiles to more negative values, indicating that these agents preferentially bind to channel inactivated states. Overall, our findings indicate that T-type Ca channels are potently blocked by a subset of neuroleptic agents and suggest that the action of these drugs on T-type Ca channels may significantly contribute to their therapeutic efficacy.

Glutamate release from rod photoreceptors is dependent on a sustained calcium influx through L-type calcium channels. Missense mutations in the CACNA1F gene in patients with incomplete X-linked congenital stationary night blindness implicate the Ca v 1.4 calcium channel subtype. Here, we describe the functional and pharmacological properties of transiently expressed human Ca v 1.4 calcium channels. Ca v 1.4 is shown to encode a dihydropyridine-sensitive calcium channel with unusually slow inactivation kinetics that are not affected by either calcium ions or by coexpression of ancillary calcium channel ␤ subunits. Additionally, the channel supports a large window current and activates near Ϫ40 mV in 2 mM external calcium, making Ca v 1.4 ideally suited for tonic calcium influx at typical photoreceptor resting potentials. Introduction of base pair changes associated with four incomplete X-linked congenital night blindness mutations showed that only the G369D alteration affected channel activation properties. Immunohistochemical analyses show that, in contrast with previous reports, Ca v 1.4 is widely distributed outside the retina, including in the immune system, thus suggesting a broader role in human physiology.

Volatile anesthetics like halothane and enflurane are of interest to clinicians and neuroscientists because of their ability to preferentially disrupt higher functions that make up the conscious state. All volatiles were once thought to act identically; if so, they should be affected equally by genetic variants. However, mutations in two distinct genes, one in Caenorhabditis and one in Drosophila, have been reported to produce much larger effects on the response to halothane than enflurane [1, 2]. To see whether this anesthesia signature is adventitious or fundamental, we have identified orthologs of each gene and determined the mutant phenotype within each species. The fly gene, narrow abdomen (na), encodes a putative ion channel whose sequence places it in a unique family; the nematode gene, unc-79, is identified here as encoding a large cytosolic protein that lacks obvious motifs. In Caenorhabditis, mutations that inactivate both of the na orthologs produce an Unc-79 phenotype; in Drosophila, mutations that inactivate the unc-79 ortholog produce an na phenotype. In each organism, studies of double mutants place the genes in the same pathway, and biochemical studies show that proteins of the UNC-79 family control NA protein levels by a posttranscriptional mechanism. Thus, the anesthetic signature reflects an evolutionarily conserved role for the na orthologs, implying its intimate involvement in drug action.

Synaptojanin is a lipid phosphatase required to degrade phosphatidylinositol 4,5 bisphosphate (PIP(2)) at cell membranes during synaptic vesicle recycling. Synaptojanin mutants in C. elegans are severely uncoordinated and are depleted of synaptic vesicles, possibly because of accumulation of PIP(2). To identify proteins that act downstream of PIP(2) during endocytosis, we screened for suppressors of synaptojanin mutants in the nematode C. elegans. A class of uncoordinated mutants called "fainters" partially suppress the locomotory, vesicle depletion, and electrophysiological defects in synaptojanin mutants. These suppressor loci include the genes for the NCA ion channels, which are homologs of the vertebrate cation leak channel NALCN, and a novel gene called unc-80. We demonstrate that unc-80 encodes a novel, but highly conserved, neuronal protein required for the proper localization of the NCA-1 and NCA-2 ion channel subunits. These data suggest that activation of the NCA ion channel in synaptojanin mutants leads to defects in recycling of synaptic vesicles.

Childhood absence epilepsy (CAE) is a type of gener-We have functionally characterized five of these mutations (F161L, E282K, C456S, V831M, and D1463N) using rat Ca v 3.2 and whole-cell patch clamp recordings in transfected HEK293 cells. Two of the mutations, F161L and E282K, mediated an ϳ10-mV hyperpolarizing shift in the half-activation potential. Mutation V831M caused a ϳ50% slowing of inactivation relative to control and shifted half-inactivation potential ϳ10 mV toward more depolarized potentials. Mean time to peak was significantly increased by mutation V831M but was unchanged for all others. No resolvable changes in the parameters of the IV relation or current kinetics were observed with the remaining mutations. The findings suggest that several of the Ca v 3.2 mutants allow for greater calcium influx during physiological activation and in the case of F161L and E282K can result in channel openings at more hyperpolarized (close to resting) potentials. This may underlie the propensity for seizures in patients with CAE.Generalized epileptic disorders involve both brain hemispheres and are characterized by abnormal synchronous electrical (electroencephalographic) activity, recorded bilaterally at seizure onset (1). Childhood absence epilepsy (CAE) 1 is a type of idiopathic generalized epilepsy and is typified by sudden brief impairment of consciousness followed by ϳ3-Hz spikeand-wave discharges (SWDs) over both brain hemispheres (2). A typical absence seizure is without convulsions and there are no reported neuropathological changes associated with this disorder (3). Spike-wave discharges in absence epilepsy involve interactions between cortical and thalamic structures (4). The classical view of SWD-based seizures, including absence epilepsy, implicates the thalamus as the site of seizure generation (5, 6). Recently, an increasing body of evidence suggests that spike-wave seizures are initiated in the neocortex and then rapidly progress to involve thalamic structures (7-9). The thalamus and cortex then engage in complex interplay that underlies SWD generation and is dependent on the activation of low voltage-activated (T-type) calcium channels (4). Indeed, reticular thalamic neurons are endowed with large T-type currents that mediate bursting behavior associated with SWDs. The critical role of T-type channels in SWD epilepsies is also supported by treatment of absence seizures using ethosuximide, an inhibitor of T-type Ca 2ϩ currents (10, 11), and by the observation that expression of these channels is increased in thalamic neurons in a genetic rat absence model (12).We now know of three genes (subtypes) encoding different types of T-type channels (Ca v 3.1, Ca v 3.2, and Ca v 3.3), all of which are subject to alternative splicing resulting in a range of different isoforms with distinct biophysical, modulatory, and pharmacological properties (13-23). It was recently shown that Ca v 3.1 knock-out mice display reduced burst mode firing activity, and that the Ca v 3.1-deficient thalamus is specifically resilie...

OBJECTIVEIn the pancreatic β-cell, ATP-sensitive K+ (KATP) channels couple metabolism with excitability and consist of Kir6.2 and SUR1 subunits encoded by KCNJ11 and ABCC8, respectively. Sulfonylureas, which inhibit the KATP channel, are used to treat type 2 diabetes. Rare activating mutations cause neonatal diabetes, whereas the common variants, E23K in KCNJ11 and S1369A in ABCC8, are in strong linkage disequilibrium, constituting a haplotype that predisposes to type 2 diabetes. To date it has not been possible to establish which of these represents the etiological variant, and functional studies are inconsistent. Furthermore, there have been no studies of the S1369A variant or the combined effect of the two on KATP channel function.RESEARCH DESIGN AND METHODSThe patch-clamp technique was used to study the nucleotide sensitivity and sulfonylurea inhibition of recombinant human KATP channels containing either the K23/A1369 or E23/S1369 variants.RESULTSATP sensitivity of the KATP channel was decreased in the K23/A1369 variant (half-maximal inhibitory concentration [IC50] = 8.0 vs. 2.5 μmol/l for the E23/S1369 variant), although there was no difference in ADP sensitivity. The K23/A1369 variant also displayed increased inhibition by gliclazide, an A-site sulfonylurea drug (IC50 = 52.7 vs. 188.7 nmol/l for the E23/S1369 variant), but not by glibenclamide (AB site) or repaglinide (B site).CONCLUSIONSOur findings indicate that the common K23/A1369 variant KATP channel displays decreased ATP inhibition that may contribute to the observed increased risk for type 2 diabetes. Moreover, the increased sensitivity of the K23/A1369 variant to the A-site sulfonylurea drug gliclazide may provide a pharmacogenomic therapeutic approach for patients with type 2 diabetes who are homozygous for both risk alleles.

OBJECTIVEThe sodium-calcium exchanger isoform 1 (NCX1) regulates cytoplasmic calcium (Ca2+c) required for insulin secretion in β-cells. NCX1 is alternatively spliced, resulting in the expression of splice variants in different tissues such as NCX1.3 and -1.7 in β-cells. As pharmacological inhibitors of NCX1 splice variants are in development, the pharmacological profile of β-cell NCX1.3 and -1.7 and the cellular effects of NCX1 inhibition were investigated.RESEARCH DESIGN AND METHODSThe patch-clamp technique was used to examine the pharmacological profile of the NCX1 inhibitor KB-R7943 on recombinant NCX1.3 and -1.7 activity. Ca2+ imaging and membrane capacitance were used to assess the effects of KB-R7943 on Ca2+c and insulin secretion in mouse and human β-cells and islets.RESULTSNCX1.3 and -1.7 calcium extrusion (forward-mode) activity was ∼16-fold more sensitive to KB-R7943 inhibition compared with cardiac NCX1.1 (IC50s = 2.9 and 2.4 vs. 43.0 μmol/l, respectively). In single mouse/human β-cells, 1 μmol/l KB-R7943 increased insulin granule exocytosis but was without effect on α-cell glucagon granule exocytosis. KB-R7943 also augmented sulfonylurea and glucose-stimulated Ca2+c levels and insulin secretion in mouse and human islets, although KB-R7943 was without effect under nonstimulated conditions.CONCLUSIONSIslet NCX1 splice variants display a markedly greater sensitivity to pharmacological inhibition than the cardiac NCX1.1 splice variant. NCX1 inhibition resulted in glucose-dependent increases in Ca2+c and insulin secretion in mouse and human islets. Thus, we identify β-cell NCX1 splice variants as targets for the development of novel glucose-sensitive insulinotropic drugs for type 2 diabetes.