We describe here several novel properties of the human ␣ 1G subunit that forms T-type calcium channels. The partial intron/exon structure of the corresponding gene CACNA1G was defined and several ␣ 1G isoforms were identified, especially two isoforms that exhibit a distinct III-IV loop: ␣ 1G-a and ␣ 1G-b . Northern blot and dot blot analyses indicated that ␣ 1G mRNA is predominantly expressed in the brain, especially in thalamus, cerebellum, and substantia nigra. Additional experiments have also provided evidence that ␣ 1G mRNA is expressed at a higher level during fetal life in nonneuronal tissues (i.e. kidney, heart, and lung). Functional expression in HEK 293 cells of a full-length cDNA encoding the shortest ␣ 1G isoform identified to date, ␣ 1G-b , resulted in transient, low threshold activated Ca 2؉ currents with the expected permeability ratio (I Sr > I Ca > I Ba ) and channel conductance (ϳ7 pS). These properties, together with slowly deactivating tail currents, are typical of those of native T-type Ca 2؉ channels. This ␣ 1G -related current was inhibited by mibefradil (IC 50 ‫؍‬ 2 M) and weakly blocked by Ni 2؉ ions (IC 50 ‫؍‬ 148 M) and amiloride (IC 50 > 1 mM). We showed that steady state activation and inactivation properties of this current can generate a "window current" in the range of ؊65 to ؊55 mV. Using neuronal action potential waveforms, we show that ␣ 1G channels produce a massive and sustained Ca 2؉ influx due to their slow deactivation properties. These latter properties would account for the specificity of Ca 2؉ influx via T-type channels that occurs in the range of physiological resting membrane potentials, differing considerably from the behavior of other Ca 2؉ channels.

The generation of the mammalian heartbeat is a complex and vital function requiring multiple and coordinated ionic channel activities. The functional role of low-voltage activated (LVA) T-type calcium channels in the pacemaker activity of the sinoatrial node (SAN) is, to date, unresolved. Here we show that disruption of the gene coding for CaV3.1/alpha1G T-type calcium channels (cacna1g) abolishes T-type calcium current (I(Ca,T)) in isolated cells from the SAN and the atrioventricular node without affecting the L-type Ca2+ current (I(Ca,L)). By using telemetric electrocardiograms on unrestrained mice and intracardiac recordings, we find that cacna1g inactivation causes bradycardia and delays atrioventricular conduction without affecting the excitability of the right atrium. Consistently, no I(Ca,T) was detected in right atrium myocytes in both wild-type and CaV3.1(-/-) mice. Furthermore, inactivation of cacna1g significantly slowed the intrinsic in vivo heart rate, prolonged the SAN recovery time, and slowed pacemaker activity of individual SAN cells through a reduction of the slope of the diastolic depolarization. Our results demonstrate that CaV3.1/T-type Ca2+ channels contribute to SAN pacemaker activity and atrioventricular conduction.

In several types of neurons, firing is an intrinsic property produced by specific classes of ion channels. Low-voltage-activated T-type calcium channels (T-channels), which activate with small membrane depolarizations, can generate burst firing and pacemaker activity. Here we have investigated the specific contribution to neuronal excitability of cloned human T-channel subunits. Using HEK-293 cells transiently transfected with the human a 1G (Ca V 3.1), a 1H (Ca V 3.2) and a 1I (Ca V 3.3) subunits, we describe significant differences among these isotypes in their biophysical properties, which are highlighted in action potential clamp studies. Firing activities occurring in cerebellar Purkinje neurons and in thalamocortical relay neurons used as voltage clamp waveforms revealed that a 1G channels and, to a lesser extent, a 1H channels produced large and transient currents, while currents related to a 1I channels exhibited facilitation and produced a sustained calcium entry associated with the depolarizing after-potential interval. Using simulations of reticular and relay thalamic neuron activities, we show that a 1I currents contributed to sustained electrical activities, while a 1G and a 1H currents generated short burst firing. Modelling experiments with the NEURON model further revealed that the a 1G channel and a 1I channel parameters best accounted for T-channel activities described in thalamocortical relay neurons and in reticular neurons, respectively. Altogether, the data provide evidence for a role of a 1I channel in pacemaker activity and further demonstrate that each T-channel pore-forming subunit displays specific gating properties that account for its unique contribution to neuronal firing.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation or the creation of derivative works without specific permission.A previously uncharacterized putative ion channel, NALCN (sodium leak channel, non-selective), has been recently shown to be responsible for the tetrodotoxin (TTX)-resistant sodium leak current implicated in the regulation of neuronal excitability. Here, we show that NALCN encodes a current that is activated by M3 muscarinic receptors (M3R) in a pancreatic b-cell line. This current is primarily permeant to sodium ions, independent of intracellular calcium stores and G proteins but dependent on Src activation, and resistant to TTX. The current is recapitulated by co-expression of NALCN and M3R in human embryonic kidney-293 cells and in Xenopus oocytes. We also show that NALCN and M3R belong to the same protein complex, involving the intracellular I-II loop of NALCN and the intracellular i3 loop of M3R. Taken together, our data show the molecular basis of a muscarinic-activated inward sodium current that is independent of G-protein activation, and provide new insights into the properties of NALCN channels.

The molecular mechanisms underlying the developmental regulation of L-type voltage-dependent Ca 2؉ channels (VDCCs) are still unknown. In this study, we have characterized the expression patterns of skeletal (␣ 1S ) and cardiac (␣ 1C ) L-type VDCCs during cardiogenic differentiation in H9C2 cells that derived from embryonic rat heart. We report that chronic treatment of H9C2 cells with 10 nM all-trans-retinoic acid (all-trans-RA) enhanced cardiac Ca 2؉ channel expression, as demonstrated by reverse transcription-polymerase chain reaction, immunoblotting, and indirect immunofluorescence studies, as well as patch-clamp experiments. In addition, RA treatment prevented expression of functional skeletal L-type VDCCs, which were restricted to myotubes that spontaneously appear in control H9C2 cultures undergoing myogenic transdifferentiation. The use of specific skeletal and cardiac markers indicated that RA, by preventing myogenic transdifferentiation, preserves cardiac differentiation of this cell line. Altogether, we provide evidence that cardiac and skeletal subtype-specific L-type Ca 2؉ channels are relevant functional markers of differentiated cardiac and skeletal myocytes, respectively. In conclusion, our data demonstrate that in vitro RA stimulates cardiac (␣ 1C ) L-type Ca 2؉ channel expression, therefore supporting the hypothesis that the RA pathway might be involved in the tissue specific expression of Ca 2؉ channels in mature cardiac cells.

Freeman-Sheldon syndrome, or distal arthrogryposis type 2A (DA2A), is an autosomal-dominant condition caused by mutations in MYH3 and characterized by multiple congenital contractures of the face and limbs and normal cognitive development. We identified a subset of five individuals who had been putatively diagnosed with "DA2A with severe neurological abnormalities" and for whom congenital contractures of the limbs and face, hypotonia, and global developmental delay had resulted in early death in three cases; this is a unique condition that we now refer to as CLIFAHDD syndrome. Exome sequencing identified missense mutations in the sodium leak channel, non-selective (NALCN) in four families affected by CLIFAHDD syndrome. We used molecular-inversion probes to screen for NALCN in a cohort of 202 distal arthrogryposis (DA)-affected individuals as well as concurrent exome sequencing of six other DA-affected individuals, thus revealing NALCN mutations in ten additional families with "atypical" forms of DA. All 14 mutations were missense variants predicted to alter amino acid residues in or near the S5 and S6 pore-forming segments of NALCN, highlighting the functional importance of these segments. In vitro functional studies demonstrated that NALCN alterations nearly abolished the expression of wild-type NALCN, suggesting that alterations that cause CLIFAHDD syndrome have a dominant-negative effect. In contrast, homozygosity for mutations in other regions of NALCN has been reported in three families affected by an autosomal-recessive condition characterized mainly by hypotonia and severe intellectual disability. Accordingly, mutations in NALCN can cause either a recessive or dominant condition characterized by varied though overlapping phenotypic features, perhaps based on the type of mutation and affected protein domain(s).

The L-type voltage-dependent calcium channel is an important link in excitation-contraction coupling of muscle cells (reviewed in refs 2 and 3). The channel has two functional characteristics: calcium permeation and receptor sites for calcium antagonists. In skeletal muscle the channel is a complex of five subunits, alpha 1, alpha 2, beta, gamma and delta. Complementary DNAs to these subunits have been cloned and their amino-acid sequences deduced. The skeletal muscle alpha 1 subunit cDNA expressed in L cells manifests as specific calcium-ion permeation, as well as sensitivity to the three classes of organic calcium-channel blockers. We report here that coexpression of the alpha 1 subunit with other subunits results in significant changes in dihydropyridine binding and gating properties. The available number of drug receptor sites increases 10-fold with an alpha 1 beta combination, whereas the affinity of the dihydropyridine binding site remains unchanged. Also, the presence of the beta subunit accelerates activation and inactivation kinetics of the calcium-channel current.

Calcium currents via low-voltage-activated T-type channels mediate burst firing, particularly in thalamic neurons. Considerable evidence supports the hypothesis that overactive T-channels may contribute to thalamocortical dysrhythmia, including absence epilepsy. Single nucleotide polymorphisms in one of the T-channel genes (CACNA1H, which encodes Ca v 3.2) are associated with childhood absence epilepsy in a Chinese population. Because only a fraction of these polymorphisms are predicted to increase channel activity and neuronal firing, we hypothesized that other channel properties may be affected. Here we describe that all the polymorphisms clustered in the intracellular loop connecting repeats I and II (I-II loop) increase the surface expression of extracellularly tagged Ca v 3.2 channels. The functional domains within the I-II loop were then mapped by deletion analysis. The first 62 amino acids of the loop (post IS6) are involved in regulating the voltage dependence of channel gating and inactivation. Similarly, the last 15 amino acids of the loop (pre IIS1) are involved in channel inactivation. In contrast, the central region of I-II loop regulates surface expression, with no significant effect on channel biophysics. Electrophysiology, luminometry, fluorescence-activated cell sorting measurements, and confocal microscopy studies demonstrate that deletion of this central region leads to enhanced surface expression of channels from intracellular compartments to the plasma membrane. These results provide novel insights into how CACNA1H polymorphisms may contribute to Ca V 3.2 channel overactivity and consequently to absence epilepsy and establish the I-II loop as an important regulator of Ca V 3.2 channel function and expression.