Structural diversity of the voltage‐dependent Ca²⁺ channel α_1E‐subunit

Abstract

Voltage-operated Ca2+ channels are heteromultimeric proteins. Their structural diversity is caused by several genes encoding homologous subunits and by alternative splicing of single transcripts. Isoforms of alpha1 subunits, which contain the ion conducting pore, have been deduced from each of the six cDNA sequences cloned so far from different species. The isoforms predicted for the alpha1E subunit are structurally related to the primary sequence of the amino terminus, the centre of the subunit (II-III loop), and the carboxy terminus. Mouse and human alpha1E transcripts have been analysed by reverse transcription-polymerase chain reaction and by sequencing of amplified fragments. For the II-III loop three different alpha1E cDNA fragments are amplified from mouse and human brain, showing that isoforms originally predicted from sequence alignment of different species are expressed in a single one. Both predicted alpha1E cDNA fragments of the carboxy terminus are identified in vivo. Two different alpha1E constructs, referring to the major structural difference in the carboxy terminus, were stably transfected in HEK293 cells. The biophysical properties of these cells were compared in order to evaluate the importance in vitro of the carboxy terminal insertion found in vivo. The wild-type alpha1E subunit showed properties, typical for a high-voltage activated Ca2+ channel. The deletion of 43 amino acid residues at the carboxy terminus does not cause significant differences in the current density and the basic biophysical properties. However, a functional difference is suggested, as in embryonic stem cells, differentiated in vitro to neuronal cells, the pattern of transcripts indicative for different alpha1E isoforms changes during development. In human cerebellum the longer alpha1E isoform is expressed predominantly. Although, it has not been possible to assign functional differences to the two alpha1E constructs tested in vitro, the expression pattern of the structurally related isoforms may have functional importance in vivo.

Cumulative inactivation of N-type CaV2.2 calcium channels modified by alternative splicing

“…A homologous exon is also present in the Ca V 2.3 gene. Its function is not known, but studies have linked it to Ca-sensitive regulation of the channel (18)(19)(20)31). Exon 18a of Ca V 2.2 is expressed abundantly in caudal regions of the mammalian nervous system and in peripheral nerve ganglia (15).…”

“…Exon 18a modifies the voltage dependence of steady-state inactivation under certain conditions without affecting the kinetics or voltage dependence of channel activation (15). A homologous alternatively spliced exon whose expression is regulated is also present in the Ca V 2.3 gene (14,17,18). Functional studies of Ca V 2.3 channels that contain this exon indicate that it regulates the Ca sensitivity of certain channel properties (19,20).…”

Immunodetection of α1E Voltage-gated Ca2+ Channel in Chromogranin-positive Muscle Cells of Rat Heart, and in Distal Tubules of Human Kidney

“…Peptide Nast-195 corresponds to a common sequence in all published ␣ 1E isoforms and peptide Nast-197 is part of a 43-amino-acid extension in the carboxy terminus of the fetal brain-derived ␣ 1Ed isoform (Schneider et al 1994;Pereverzev et al 1998), which is also located in two other deduced longer ␣ 1E isoforms, ␣ 1Ee and ␣ 1Ef . The peptides (a) Nast-195, SGILEGFDPPHPCGV-QGC (aa 256-273, in the loop IS5 to pore region; GenBank L27745), and (b) Nast-197 GIYLPSDTQEHAG[C] (aa 1981, in the carboxy terminus) were synthesized by the solid-phase method (Merrifield 1986).…”

“…Fragments of cDNA were amplified by PCR (PerkinElmer Cetus Instruments; Norwalk, CT) after reverse transcription of total RNA using standard protocols as specified (Schneider et al 1994;Pereverzev et al 1998). The sequences of primer pairs and the annealing temperatures used for the PCR reactions of ␣ 1 subunits from high-voltage-activated Ca 2 ϩ channels and for the housekeeping enzyme hypoxanthine phosphoribosyltransferase have been summarized recently .…”

“…Microsomal membrane proteins from untransfected and stably transfected HEK-293 cells and from the rat insulinoma cell line INS-1 (Asfari et al 1992) were isolated according to Pereverzev et al (1998). The rat myoblast cell line H9c2 was grown in DMEM supplemented with 2 mM l -glutamine, 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 100 g/ml streptomycin.…”