Distinctive modulatory effects of five human auxiliary beta2 subunit splice variants on L-type calcium channel gating.
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.
Takahashi
Shoji X
SX
Calcium Signals Laboratory, Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Mittman
Scott
S
Colecraft
Henry M
HM
D000222:Adaptation, Physiological; D001921:Brain / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000145:classification* / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D014644:Genetic Variation; D006706:Homeostasis / Q000502:physiology; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007668:Kidney / Q000196:embryology / Q000502:physiology; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D009154:Mutation; D009206:Myocardium / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000502:physiology; D011994:Recombinant Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
C107597:CACNB2 protein, human; D020746:Calcium Channels, L-Type; D021122:Protein Subunits; D011994:Recombinant Proteins