Primary structure of a calcium channel that is highly expressed in the rat cerebellum.

Abstract

Previous molecular cloning experiments showed that multiple isoforms of the al subunit of voltagegated Ca channels are expressed in the mammalian brain (designated rbA, rbB, rbC, and rbD). We report here the isolation and characterization of cDNAs encoding the rat brain class A (rbA) Ca channel. The rbA-126 cDNA encodes a 2212-amino acid protein that shares 33% sequence identity with the al subunits of cardiac and skeletal muscle dihydropyridine-sensitive Ca channels. When compared with other Ca channels, the rbA channel is notably different in both the carboxyl terminus and in a large (474-amino acid) hydrophilic segment between domains II and HI. Northern blot analysis shows that rbA transcripts are expressed in all regions of the rat central nervous system, but most prominently in the cerebellum. A more widespread distribution of rbA Ca channels is indicated by PCR analysis, which demonstrates the presence of class A transcripts in the rat heart and pituitary but not in the spleen, kidney, or liver. The rbA cDNA appears to encode a Ca channel al subunit that is distinct from the dihydropyridine-sensitive Ca channel sequences and that is expressed in a variety of excitable cells.Voltage-gated Ca channels form a diverse set of molecules that mediate Ca entry into a wide variety ofcell types. In neurons, Ca entry both contributes to the electrical properties of cells and affects the level of intracellular Ca, which acts as a second messenger (for reviews see refs. 1 and 2). On the basis of electrophysiological and pharmacological criteria, four major types ofCa channels have been described in neurons (designated T, L, N, and P; reviewed in ref.3). However, Ca channels from different neuronal cell types often exhibit pharmacological and electrophysiological profiles that do not correlate exactly with any one of the four types, which suggests that additional Ca channel diversity exists (3,4,39).Detailed biochemical studies of the dihydropyridine (DHP)-sensitive Ca channel from skeletal muscle T-tubule membranes (L-type Ca channel) have shown that it is composed of five distinct subunits (al, a2, ,, y, and 8; reviewed in refs. 5 and 6). Molecular cloning has revealed that the al subunit is a 212-kDa protein (7) that is structurally homologous to the a subunit of voltage-gated Na channels (8,9). Both the Ca channel al subunit and the Na channel a subunit contain four repeated domains, each containing six to eight putative transmembrane segments. More recently, fulllength cDNAs for the al subunits of cardiac and smooth muscle L-type Ca channels have also been reported (10-12). Expression of these cloned al-subunit cDNAs in a number of test systems shows that this subunit encodes a voltage-gated, 11,13).Utilizing low-stringency hybridization with a skeletal muscle probe, Snutch et al. (14) isolated a family of cDNAs from rat brain that are related to the al subunits of L-type Ca channels. With these probes we have begun to address a number of basic questions concerning the structure, expression, and regulation...

Molecular determinants of inactivation and G protein modulation in the intracellular loop connecting domains I and II of the calcium channel α 1A subunit

“…cDNAs encoding Ca 2ϩ channel subunits ␣ 1A [rbA isoform, (19)] and ␤ 1b were cloned in pMT2XS (8), ␣ 1C [rbC-II isoform (18)] in pZem229, ␣ 2 ␦ (21) in pZEM228, and CD8 in EBPpcD. tsA-201 cells were transfected with the ␣ 1 , ␣ 2 ␦, and ␤ cDNAs in 1:1:1 molar ratios plus CD8 in either calcium phosphate or lipofectamine (Stratagene) and incubated for at least 48 h. Positively transfected cells were identified by labeling with anti-CD8 antibody tagged with fluorophore and analyzed by whole-cell patch clamp as described (14,22).…”

“…A related consensus sequence is within or adjacent to the region of the COOH terminus of inwardly rectifying K ϩ channels which is implicated in binding and regulation by G␤␥ subunits (16,17). It is also found in the intracellular loops connecting domains I and II of ␣ 1A and ␣ 1B , which are modulated by G␤␥, but not in the corresponding segment of ␣ 1C , which is not modulated by this pathway (18)(19)(20). Thus, this consensus sequence could be involved in G protein modulation of Ca 2ϩ channels.…”

Migraine, ataxia and epilepsy: a challenging spectrum of genetically determined calcium channelopathies

“…33 Mutations in the P/Q Type Calcium Channel α 1A Subunit Gene in FHM Using exon trapping, a human cDNA highly homologous to a brain-specific rabbit and rat voltage gated P/Q type calcium channel α 1A subunit gene was identified. [34][35][36] The human gene was designated CACNL1A4, 37 but according to a newly proposed nomenclature the gene is called CACNA1A. 29 The gene is transcribed specifically in cerebellum, cerebral cortex, thalamus and hypothalamus.…”

Effects of familial hemiplegic migraine type 1 mutations on neuronal P/Q-type Ca 2+ channel activity and inhibitory synaptic transmission

“…The hypothesis that migraine may be a synaptic disease has gained further credence through pioneering studies of familial hemiplegic migraine type 1 (FHM1), a rare hereditary form of migraine, and the identification of mutations in a neuronal Ca 2ϩ channel vital for neurotransmission (4). The affected CACNA1A gene encodes the pore-forming ␣ 1A subunit of the P͞Q-type channel (Ca V 2.1), a predominant mediator of voltage-gated Ca 2ϩ entry and transmitter release at many synapses in the central nervous system (5)(6)(7)(8)(9). Alterations in synaptic signaling are of likely importance for cross-talk between an ascending pain transmission pathway and inhibition by a powerful descending modulatory system, interactions that ultimately govern the generation of headache pain (10,11).…”