RESUMO
Voltage-dependent calcium channel (Ca(v)) pores are modulated by cytosolic beta subunits. Four beta-subunit genes and their splice variants offer a wide structural array for tissue- or disease-specific biophysical gating phenotypes. For instance, the length of the N terminus of beta(2) subunits has major effects on activation and inactivation rates. We tested whether a similar mechanism principally operates in a beta(1) subunit. Wild-type beta(1a) subunit (N terminus length 60 aa) and its newly generated N-terminal deletion mutants (51, 27 and 18 aa) were examined within recombinant L-type calcium channel complexes (Ca(v)1.2 and alpha(2)delta2) in HEK293 cells at the whole-cell and single-channel level. Whole-cell currents were enhanced by co-transfection of the full-length beta(1a) subunit and by all truncated constructs. Voltage dependence of steady-state activation and inactivation did not depend on N terminus length, but inactivation rate was diminished by N terminus truncation. This was confirmed at the single-channel level, using ensemble average currents. Additionally, gating properties were estimated by Markov modeling. In confirmation of the descriptive analysis, inactivation rate, but none of the other transition rates, was reduced by shortening of the beta(1a) subunit N terminus. Our study shows that the length-dependent mechanism of modulating inactivation kinetics of beta(2) calcium channel subunits can be confirmed and extended to the beta(1) calcium channel subunit.
Assuntos
Canais de Cálcio Tipo L , Ativação do Canal Iônico/fisiologia , Mutação , Isoformas de Proteínas , Subunidades Proteicas , Processamento Alternativo , Sequência de Aminoácidos , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Humanos , Cadeias de Markov , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
L-type calcium channels are composed of a pore, alpha1c (Ca(V)1.2), and accessory beta- and alpha2delta-subunits. The beta-subunit core structure was recently resolved at high resolution, providing important information on many functional aspects of channel modulation. In this study we reveal differential novel effects of five beta2-subunits isoforms expressed in human heart (beta(2a-e)) on the single L-type calcium channel current. These splice variants differ only by amino-terminal length and amino acid composition. Single-channel modulation by beta2-subunit isoforms was investigated in HEK293 cells expressing the recombinant L-type ion conducting pore. All beta2-subunits increased open probability, availability, and peak current with a highly consistent rank order (beta2a approximately = beta2b > beta2e approximately = beta2c > beta2d). We show graded modulation of some transition rates within and between deep-closed and inactivated states. The extent of modulation correlates strongly with the length of amino-terminal domains. Two mutant beta2-subunits that imitate the natural span related to length confirm this conclusion. The data show that the length of amino termini is a relevant physiological mechanism for channel closure and inactivation, and that natural alternative splicing exploits this principle for modulation of the gating properties of calcium channels.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Miocárdio/química , Adulto , Sequência de Bases , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Linhagem Celular , Clonagem Molecular , Primers do DNA , Expressão Gênica , Humanos , Ativação do Canal Iônico , Masculino , Splicing de RNA , Processos EstocásticosRESUMO
Overexpression of human cardiac L-type Ca(2+) channel pores (hCa(v)1.2) in mice causes heart failure. Earlier studies showed Ca(v)1.2-mRNA increase by 2.8-fold, but whole-cell current density enhancement by