P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mammalian nervous system. The alpha 1A calcium channel has been implicated in the etiologies of conditions such as episodic ataxia, epilepsy and familial migraine, and shares several properties with native P- and Q-type channels. However, the exact relationship between alpha 1A and P- and Q-type channels is unknown. Here we report that alternative splicing of the alpha 1A subunit gene results in channels with distinct kinetic, pharmacological and modulatory properties. Overall, the results indicate that alternative splicing of the alpha 1A gene generates P-type and Q-type channels as well as multiple phenotypic variants.
Analgesic therapies are still limited and sometimes poorly effective, therefore finding new targets for the development of innovative drugs is urgently needed. In order to validate the potential utility of blocking T-type calcium channels to reduce nociception, we explored the effects of intrathecally administered oligodeoxynucleotide antisenses, specific to the recently identified T-type calcium channel family (Ca V 3.1, Ca V 3.2, and Ca V 3.3), on reactions to noxious stimuli in healthy and mononeuropathic rats. Our results demonstrate that the antisense targeting Ca V 3.2 induced a knockdown of the Ca V 3.2 mRNA and protein expression as well as a large reduction of 'Ca V 3.2-like' T-type currents in nociceptive dorsal root ganglion neurons. Concomitantly, the antisense treatment resulted in major antinociceptive, anti-hyperalgesic, and anti-allodynic effects, suggesting that Ca V 3.2 plays a major pronociceptive role in acute and chronic pain states. Taken together, the results provide direct evidence linking Ca V 3.2 T-type channels to pain perception and suggest that Ca V 3.2 may offer a specific molecular target for the treatment of pain.
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.
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.
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).
At least three genes encode T-type calcium channel alpha(1) subunits, and identification of cDNA transcripts provided evidence that molecular diversity of these channels can be further enhanced by alternative splicing mechanisms, especially for the alpha(1G) subunit (Ca(V)3.1). Using whole-cell patch-clamp procedures, we have investigated the electrophysiological properties of five isoforms of the human alpha(1G) subunit that display a distinct III-IV linker, namely, alpha(1G-a), alpha(1G-b), and alpha(1G-bc), as well as a distinct II-III linker, namely, alpha(1G-ae), alpha(1G-be), as expressed in HEK-293 cells. We report that insertion e within the II-III linker specifically modulates inactivation, steady-state kinetics, and modestly recovery from inactivation, whereas alternative splicing within the III-IV linker affects preferentially kinetics and voltage dependence of activation, as well as deactivation and inactivation. By using voltage-clamp protocols mimicking neuronal activities, such as cerebellar train of action potentials and thalamic low-threshold spike, we describe that inactivation properties of alpha(1G-a) and alpha(1G-ae) isoforms can support channel behaviors reminiscent to those described in native neurons. Altogether, these data demonstrate that expression of distinct variants for the T-type alpha(1G) subunit can account for specific low-voltage-activated currents observed in neuronal tissues.
We have cloned and expressed a human ␣ 1I subunit that encodes a subtype of T-type calcium channels. The predicted protein is 95% homologous to its rat counterpart but has a distinct COOH-terminal region. Its mRNA is detected almost exclusively in the human brain, as well as in adrenal and thyroid glands. Calcium currents generated by the functional expression of human ␣ 1I and ␣ 1G subunits in HEK-293 cells were compared. The ␣ 1I current activated and inactivated ϳ10 mV more positively. Activation and inactivation kinetics were up to six times slower, while deactivation kinetics was faster and showed little voltage dependence. A slower recovery from inactivation, a lower sensitivity to Ni 2؉ ions (IC 50 ϳ180 M), and a larger channel conductance (ϳ11 picosiemens) were the other discriminative features of the ␣ 1I current. These data demonstrate that the ␣ 1I subunit encodes T-type Ca 2؉ channels functionally distinct from those generated by the human ␣ 1G or ␣ 1H subunits and point out that human and rat ␣ 1I subunits have species-specific properties not only in their primary sequence, but also in their expression profile and electrophysiological behavior.Voltage-dependent calcium channels control the rapid entry of Ca 2ϩ ions into a wide variety of cell types and are therefore involved in both electrical and cellular signaling. Electrophysiological studies have identified two major Ca 2ϩ channel types as high voltage-activated and low voltage-activated channels (1, 2) with this latter class being also identified as T-type Ca 2ϩ channels (3). T-type Ca 2ϩ channels were originally defined by their activation at low membrane potential, their fast time course, and their small single channel conductance (4, 5). These channels have been identified on a large variety of neurons, and it has become obvious that significant functional diversity exists in the gating behavior of T-type channels, particularly in inactivation kinetics, voltage dependence of steady-state inactivation, and pharmacology (6). The recent identification of several novel genes encoding a subset of homologous Ca 2ϩ channel ␣ 1 subunits, e.g. the ␣ 1G subunit (7-9), the ␣ 1H subunit (10,11), and the rat ␣ 1I subunit (12), has revealed that diversity of T-type voltage-dependent calcium channels is primarily related to the expression of distinct ␣ 1 subunits. Indeed, the expression of the various ␣ 1G and ␣ 1H subunits (7-12) produces Ca 2ϩ currents with the typical signature of T-type channels but with specific features, such as the block by Ni 2ϩ , which discriminates between ␣ 1G and ␣ 1H currents (13). By contrast, the biophysical properties of T-type channels generated by the ␣ 1I subunit markedly differ from those made of ␣ 1G and ␣ 1H subunits (12,14,15), and it was postulated that the ␣ 1I subunit is responsible for native "slow" T-type currents observed in rat thalamic neurons (16). To date the ␣ 1I subunit has only been cloned from rat (12) and whether the ␣ 1I subunit encodes an atypical T-type Ca 2ϩ channel certainly needs further investig...
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