Alternative Splicing of the β₄Subunit Has α₁Subunit Subtype-Specific Effects on Ca²⁺Channel Gating

Abstract

Ca 2ϩ channel ␤ subunits are important molecular determinants of the kinetics and voltage dependence of Ca 2ϩ channel gating. Through direct interactions with channel-forming ␣ 1 subunits, ␤ subunits enhance expression levels, accelerate activation, and have variable effects on inactivation. Four distinct ␤ subunit genes each encode five homologous sequence domains (D1-5), three of which (D1, D3, and D5) undergo alternative splicing. We have isolated from human spinal cord a novel alternatively spliced ␤ 4 subunit containing a short form of domain D1 (␤ 4a ) that is highly homologous to N termini of Xenopus and rat ␤ 3 subunits. The purpose of this study was to compare the gating properties of various ␣ 1 subunit complexes containing ␤ 4a with those of complexes containing a ␤ 4 subunit with a longer form of domain D1, ␤ 4b . Expression in Xenopus oocytes revealed that, relative to ␣ 1A and ␣ 1B complexes containing ␤ 4b , the voltage dependence of activation and inactivation of complexes containing ␤ 4a were shifted to more depolarized potentials. Moreover, ␣ 1A and ␣ 1B complexes containing ␤ 4a inactivated at a faster rate. Interestingly, ␤ 4 subunit alternative splicing did not influence the gating properties of ␣ 1C and ␣ 1E subunits. Experiments with ␤ 4 deletion mutants revealed that both the N and C termini of the ␤ 4 subunit play critical roles in setting voltage-dependent gating parameters and that their effects are ␣ 1 subunit specific. Our data are best explained by a model in which distinct modes of activation and inactivation result from ␤-subunit splice variant-specific interactions with an ␣ 1 subunit gating structure. Key words: ␤4 subunit; alternative splicing; N terminus; calcium channel; gating; voltage clamp; spinal cordNeuronal high voltage-activated Ca 2ϩ channels (L, N, P/Q, and R) consist of at least four subunits, ␣ 1 , ␣ 2 /␦, and ␤ , with a fifth subunit, ␥, being recently described (Letts et al., 1998). Different Ca 2ϩ channel phenotypes arise primarily from the expression of five unique ␣ 1 subunit genes (␣ 1A -␣ 1E ). These genes encode large pore-forming proteins (Ͼ2200 amino acids) that are differentially distributed throughout the nervous system (Westenbroek et al., 1990(Westenbroek et al., , 1998. Synaptic N-, P/Q-, and R-type channels, formed by ␣ 1B , ␣ 1A , and ␣ 1E subunits, respectively, play a principal role in regulating neurotransmitter release (Turner et al., 1992;Takahashi and Momiyama, 1993;Wheeler et al., 1994;Wu et al., 1999).Ca 2ϩ channel ␤ subunits (subtypes 1-4) are highly homologous intracellular proteins with primary sequences ranging from 480 to 630 amino acids (for review, see Birnbaumer et al., 1998). The sequence can be divided into five domains on the basis of the regions of amino acid identity between subtypes. All ␤ subunits contain a highly conserved ␤ interaction domain (BID) in domain 4, which has been shown to interact with high affinity to an ␣ interaction domain (AID) on the I-II linker of ␣ 1 subunits (Pragnell et al., 1994;De Waard and Campbell,...

Direct interaction with a nuclear protein and regulation of gene silencing by a variant of the Ca 2+ -channel β 4 subunit

“…4). Three of these, clones ␤8-1, ␤11-1, and ␤13-1, are highly homologous to the human ␤ 4b and ␤ 4a isoforms reported previously (18,19). The cochlear clone ␤E-1 and the brain clone ␤9-2, however, represent splice variants of ␤ 4 of which we know no previous report.…”

Ca 2+ channel-independent requirement for MAGUK family CACNB4 genes in initiation of zebrafish epiboly

“…The number and size of exons comprising both ␤4 loci are similar to the human gene. As in humans, both ␤4.1 and ␤4.2 undergo Nterminal alternative splicing to form ␤4a and ␤4b isoforms (14). The zebrafish ␤ subunit SH3 and GK domains are 95-96% identical to human, with the majority of amino acid substitutions being conservative (Fig.…”

Non-voltage-gated L-type Ca2+ Channels in Human T Cells

“…These primers detect both known splice variants, namely ␤4a and ␤4b. These splice variants differ at their N termini (42). Two PCR products were detected in all of the T cell lines examined (Fig.…”