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.

Sequence analysis of the human genome permitted cloning of five Ca(2+)-channel beta(2) splice variants (beta(2a)-beta(2e)) that differed only in their proximal amino-termini. The functional consequences of such beta(2)-subunit diversity were explored in recombinant L-type channels reconstituted in HEK 293 cells. Beta(2a) and beta(2e) targeted autonomously to the plasma membrane, whereas beta(2b)-beta(2d) localized to the cytosol when expressed in HEK 293 cells. The pattern of modulation of L-type channel voltage-dependent inactivation gating correlated with the subcellular localization of the component beta(2) variant-membrane-bound beta(2a) and beta(2e) subunits conferred slow(er) channel inactivation kinetics and displayed a smaller fraction of channels recovering from inactivation with fast kinetics, compared to beta(2b)-beta(2d) channels. The varying effects of beta(2) subunits on inactivation gating were accounted for by a quantitative model in which L-type channels reversibly distributed between fast and slow forms of voltage-dependent inactivation-membrane-bound beta(2) subunits substantially decreased the steady-state fraction of fast inactivating channels. Finally, the beta(2) variants also had distinctive effects on L-type channel steady-state activation gating, as revealed by differences in the waveforms of tail-activation (G-V) curves, and conferred differing degrees of prepulse facilitation to the channel. Our results predict important physiological consequences arising from subtle changes in Ca(2+)-channel beta(2)-subunit structure due to alternative splicing and emphasize the utility of splice variants in probing structure-function mechanisms.

High-voltage-activated Ca 2؉ channels regulate diverse functions ranging from muscle contraction to synaptic transmission. Association between auxiliary ␤-and distinct pore-forming ␣1-subunits is obligatory for forming functional high-voltage-activated Ca 2؉ channels, yet the structural determinants underlying this interaction remain poorly understood. Recently, homology modeling of Ca 2؉ -channel ␤1b-subunit identified src homology 3 (SH3) and guanylate kinase (GK) motifs in a tandem arrangement reminiscent of the membrane-associated guanylate kinase (MAGUK) class of scaffolding proteins. However, direct evidence for MAGUK-like properties and their functional implications in ␤-subunits is lacking. Here, we show a functional requirement for both SH3 and GK domains in ␤2a. Point mutations in either the putative ␤2a SH3 or GK domains severely blunted modulation of recombinant L-type channels, showing the importance of both motifs for a functional ␣1-␤ interaction. Coexpression of these functionally deficient ␤2a-SH3 and GK mutants rescued WT currents, demonstrating trans complementation similar to that observed in MAGUKs. Truncated ''hemi-␤2a'' subunits, containing either the SH3 or GK domain, were ineffective on their own, but reconstituted WT currents when coexpressed. Moreover, the SH3 and GK domains were found to interact in vitro. These findings reveal MAGUK-like properties in ␤-subunits that are critical for ␣1-subunit modulation, revise current models of ␣1-␤ association, and predict new physiological dimensions of ␤-subunit function. fluxes that drive diverse biological processes such as muscle contraction, synaptic transmission, hormone secretion, and gene expression (1-3). Structurally, HVA Ca 2ϩ channels are heteromultimeric proteins composed of pore-forming ␣ 1 -subunits combined with auxiliary ␤-, ␣ 2 ␦-, and sometimes ␥-subunits. Seven genes encoding ␣ 1 -subunits (Ca V 1.1-1.4, Ca V 2.1-2.3), four encoding ␤-subunits (␤ 1 -␤ 4 ), and three encodingAmong the auxiliary proteins, ␤-subunits play a crucial role in the formation and behavior of all functional HVA Ca 2ϩ channels. Association with ␤s is required for efficient membrane trafficking of ␣ 1 (4-6), increasing channel open probability (P o ) (7), and normalizing the voltage dependence of channel activation (8, 9). The physiological importance of ␤-subunits is demonstrated by the severe phenotypes of knockout mice: ␤ 1 knockout is lethal at birth because of asphyxiation (10); ␤ 2a knockout is embryonic lethal due to cardiac defects (11); and ␤ 4 knockout causes the lethargic epileptic phenotype (12). Therefore, in-depth understanding of how ␤s modulate ␣ 1 is critical for insights into the operation of HVA Ca 2ϩ channels in both normal and disease states.Primary sequence alignments indicate a modular domain structure for Ca 2ϩ -channel ␤-subunits (see Fig. 2 A): three variable regions susceptible to alternative splicing (domains D1, D3, and D5) are separated by two highly conserved domains (D2 and D4) (13-15). This design suggests a dichotomous i...

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Auxiliary Ca 2 ϩ channel ␤ subunits (Ca V ␤ ) regulate cellular Ca 2 ϩ signaling by trafficking pore-forming ␣ 1 subunits to the membrane and normalizing channel gating. These effects are mediated through a characteristic src homology 3/ guanylate kinase (SH3-GK) structural module, a design feature shared in common with the membrane-associated guanylate kinase (MAGUK) family of scaffold proteins. However, the mechanisms by which the Ca V ␤ SH3-GK module regulates multiple Ca 2 ϩ channel functions are not well understood. Here, using a split-domain approach, we investigated the role of the interrelationship between Ca V ␤ SH3 and GK domains in defining channel properties. The studies build upon a previously identified split-domain pair that displays a trans SH3-GK interaction, and fully reconstitutes Ca V ␤ effects on channel trafficking, activation gating, and increased open probability ( P o ). Here, by varying the precise locations used to separate SH3 and GK domains and monitoring subsequent SH3-GK interactions by fluorescence resonance energy transfer (FRET), we identified a particular split-domain pair that displayed a subtly altered configuration of the trans SH3-GK interaction. Remarkably, this pair discriminated between Ca V ␤ trafficking and gating properties: ␣ 1C targeting to the membrane was fully reconstituted, whereas shifts in activation gating and increased P o functions were selectively lost. A more extreme case, in which the trans SH3-GK interaction was selectively ablated, yielded a split-domain pair that could reconstitute neither the trafficking nor gating-modulation functions, even though both moieties could independently engage their respective binding sites on the ␣ 1C (Ca V 1.2) subunit. The results reveal that Ca V ␤ SH3 and GK domains function codependently to tune Ca 2 ϩ channel trafficking and gating properties, and suggest new paradigms for physiological and therapeutic regulation of Ca 2 ϩ channel activity.

Voltage-dependent calcium-channel β subunits (Ca V β) strongly modulate pore-forming α 1 subunits by trafficking channel complexes to the plasma membrane and enhancing channel open probability (P o ). Despite their central role, it is unclear whether binding of a single Ca V β, or multiple Ca V βs, to an α 1 subunit governs the two distinct functions. Conventional experiments utilizing coexpression of α 1 and Ca V β subunits have been unable to resolve the ambiguity due to difficulties in establishing their stoichiometry in functional channels. Here, we unambiguously establish a 1 : 1 stoichiometry by covalently linking Ca V β 2b to the carboxyl terminus of α 1C (Ca V 1.2), creating α 1C ·β 2b . Recombinant L-type channels reconstituted in HEK 293 cells with α 1C ·β 2b supported whole-cell currents to the same extent as channels reconstituted via coexpression of the individual subunits. Analysis of gating charge showed α 1C ·β 2b fully restored channel trafficking to the plasma membrane. Co-transfecting Ca V β 2a with α 1C ·β 2b had little further impact on function. To rule out the possibility that fused Ca V β 2b was interacting in trans with neighbouring α 1 molecules, α 1C ·β 2b was cotransfected with α 1B (Ca V 2.2), and pharmacological block with nimodipine showed an absence of α 1B trafficking. These results establish that association of a single Ca V β with a pore-forming α 1 subunit captures the functional essence of HVA calcium channels, and introduce α 1 -Ca V β fusion proteins as a powerful new tool to probe structure-function mechanisms.