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.

P/Q-type (Ca v 2.1) calcium channels support a host of Ca 2ϩ -driven neuronal functions in the mammalian brain. Alternative splicing of the main ␣ 1A (␣ 1 2.1) subunit of these channels may thereby represent a rich strategy for tuning the functional profile of diverse neurobiological processes. Here, we applied a recently developed "transcript-scanning" method for systematic determination of splice variant transcripts of the human ␣ 1 2.1 gene. This screen identified seven loci of variation, which together have never been fully defined in humans. Genomic sequence analysis clarified the splicing mechanisms underlying the observed variation. Electrophysiological characterization and a novel analytical paradigm, termed strength-current analysis, revealed that one focus of variation, involving combinatorial inclusion and exclusion of exons 43 and 44, exerted a primary effect on current amplitude and a corollary effect on Ca 2ϩ -dependent channel inactivation. These findings significantly expand the anticipated scope of functional diversity produced by splice variation of P/Q-type channels.

SUMMARY1. Cells in the ganglion cell layer of salamander retinal slices were voltage clamped using patch pipettes. Light elicited transient excitatory postsynaptic currents (EPSCs) in on-off ganglion cells and sustained EPSCs in on ganglion cells. Lightevoked inhibitory postsynaptic currents in these cells could be blocked by 100 /LMbicuculline methobromide and 500 nM-strychnine.2. In the presence of external Cd2", at a concentration that blocked light-evoked synaptic inputs, NV-methyl-D-aspartate (NMDA) and the non-NMDA-receptor agonists, quisqualate and kainate, gated conductances in both on-off and on ganglion cells. The current-voltage (I-V) curve for the conductance elicited by NMDA had a negative slope between -40 and -70 mV and a reversal potential near 0 mV. The I-V curves for the non-NMDA-receptor-mediated conductances were nearly linear and also had reversal potentials near 0 mV.3. I-V curves were measured at an early time point near the peak of transient EPSCs and at a later time point during the decay phase of the responses. The late I-V curve had a negative slope below -40 mV. The early I-V curve had a positive slope over the entire voltage range but the slope was greater at positive than at negative potentials. The evoked current reversed near 0 mV at both time points.4. The region of negative slope of the late I-V curve was eliminated when Mg2" was removed from the external saline. A slowly decaying component of transient EPSCs was eliminated in 20 /tM-DL-2-amino-7-phosphonoheptanoate (AP7), an NMDAreceptor antagonist.5. Application of 1 /aM-6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA-receptor antagonist at this concentration, blocked a fast component of transient EPSCs.6 S. MITTMAN, W. R. TA YLOR AND D. R. COPENHAGEN n = 5), and a faster non-NMDA-receptor-mediated component having a time-topeak of 28 + 10 ms and an e-fold decay time of 43 + 20 ms at -31 mV (n = 8).7. A similar analysis of sustained EPSCs of on ganglion cells showed that these currents resulted from sustained activation of both NMDA and non-NMDA receptors.

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.