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Distinctive Modulatory Effects of Five Human Auxiliary ß₂ Subunit Splice Variants on L-Type Calcium Channel Gating

Shoji X. Takahashi ^*, Scott Mittman  and Henry M. Colecraft ^*

Calcium Signals Laboratory, ^* Department of Biomedical Engineering, Department of Anesthesiology, Johns Hopkins University School of Medicine, Baltimore, Maryland

Correspondence: Address reprint requests to Henry M. Colecraft, Calcium Signals Laboratory, Dept. of Biomedical Engineering, Johns Hopkins University School of Medicine, 720 Rutland Ave., 726 Traylor Bldg., Baltimore, MD 21205. Tel.: 410-955-0072; Fax: 410-614-8269; E-mail: hcolecra{at}bme.jhu.edu.

Sequence analysis of the human genome permitted cloning of five Ca²⁺-channel ß₂ splice variants (ß_2a–ß_2e) that differed only in their proximal amino-termini. The functional consequences of such ß₂-subunit diversity were explored in recombinant L-type channels reconstituted in HEK 293 cells. ß_2a and ß_2e targeted autonomously to the plasma membrane, whereas ß_2b–ß_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 ß₂ variant—membrane-bound ß_2a and ß_2e subunits conferred slow(er) channel inactivation kinetics and displayed a smaller fraction of channels recovering from inactivation with fast kinetics, compared to ß_2b–ß_2d channels. The varying effects of ß₂ 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 ß₂ subunits substantially decreased the steady-state fraction of fast inactivating channels. Finally, the ß₂ 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 Ca²⁺-channel ß₂-subunit structure due to alternative splicing and emphasize the utility of splice variants in probing structure-function mechanisms.

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