Novel functional properties of Ca²⁺ channel β subunits revealed by their expression in adult rat heart cells

Abstract

Recombinant adenoviruses were used to overexpress green fluorescent protein (GFP)‐fused auxiliary Ca2+ channel β subunits (β1‐β4) in cultured adult rat heart cells, to explore new dimensions of β subunit functions in vivo. Distinct β‐GFP subunits distributed differentially between the surface sarcolemma, transverse elements, and nucleus in single heart cells. All β‐GFP subunits increased the native cardiac whole‐cell L‐type Ca2+ channel current density, but produced distinctive effects on channel inactivation kinetics. The degree of enhancement of whole‐cell current density was non‐uniform between β subunits, with a rank order of potency β2aαβ4 > β1b > β3. For each β subunit, the increase in L‐type current density was accompanied by a correlative increase in the maximal gating charge (Qmax) moved with depolarization. However, β subunits produced characteristic effects on single L‐type channel gating, resulting in divergent effects on channel open probability (Po). Quantitative analysis and modelling of single‐channel data provided a kinetic signature for each channel type. Spurred on by ambiguities regarding the molecular identity of the actual endogenous cardiac L‐type channel β subunit, we cloned a new rat β2 splice variant, β2b, from heart using 5′ rapid amplification of cDNA ends (RACE) PCR. By contrast with β2a, expression of β2b in heart cells yielded channels with a microscopic gating signature virtually identical to that of native unmodified channels. Our results provide novel insights into β subunit functions that are unattainable in traditional heterologous expression studies, and also provide new perspectives on the molecular identity of the β subunit component of cardiac L‐type Ca2+ channels. Overall, the work establishes a powerful experimental paradigm to explore novel functions of ion channel subunits in their native environments.

Reverse engineering the L-type Ca2+ channel α1c subunit in adult cardiac myocytes using novel adenoviral vectors

“…Cultured adult guinea pig cardiomyocytes typically exhibited large I CaL densities in the presence of β 2a -GFP, consistent with previous reports [24,26,27]. However, cells transduced with α 1c-D-, in addition to β 2a -GFP, showed a larger I CaL density than those infected with β 2a -GFP alone (Fig.…”

“…In these experiments, we co-expressed the rat β 2a subunit (fused to GFP), as it has previously been demonstrated that β subunit coexpression is a limiting factor for L-type Ca 2+ current density in cultured adult cardiomyocytes [24,26,27]. In preliminary experiments, we also observed that I CaL declined markedly over 48 h in culture in the absence of β subunit over-expression (data not shown).…”

“…To assess the ability of these novel adenoviruses to form viable L-type Ca 2+ channels, we coexpressed in A549 cells, either α 1c-wt or α 1c-D-, with a β 2a -GFP adenovirus [24], and an ecdysone-receptor adenovirus, AdDMEcR [25]. Both α 1c-wt and α 1c-D-adenoviruses formed functional L-type Ca 2+ channels in A549 cells (Figs.…”

Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations

“…The G402S and G406R mutations were introduced by sitedirected mutagenesis using Quikchange (Stratagene). Ca V ␤ 2b and Ca V ␣ 2 ␦ 1 are the accessory subunits associated with Ca V 1.2 in the human heart (7,8). The rabbit Ca V ␤ 2b and Ca V ␣ 2 ␦ 1 clones used for expression in Xenopus oocytes were a kind gift from N. Dascal (Tel Aviv University, Ramat Aviv, Israel).…”

β-Adrenergic regulation requires direct anchoring of PKA to cardiac Ca V 1.2 channels via a leucine zipper interaction with A kinase-anchoring protein 15

“…Voltage-gated L-type Ca 2ϩ channels are multisubunit complexes composed of a poreforming ␣ 1 subunit and auxiliary ␤ and ␣ 2 ␦ subunits (4). In cardiac muscle, a distinct ␣ 1 subunit (␣ 1 1.2a) (5,6), an ␣ 2 ␦ subunit (7), and several isoforms of ␤ subunits [␤ 1b and ␤ 2a-d (8)(9)(10)(11)] have been identified and implicated to form the Ca V 1.2 channel. As for the skeletal muscle Ca V 1.1 channel (12,13), two size forms of the ␣ 1 1.2a subunit of Ca V 1.2 channels, Ϸ240 and 210 kDa, are present in cardiac muscle and differ by truncation at the C terminus (14).…”