Functional characterization of the L-type Ca2+ channel Cav1.4alpha1 from mouse retina

Abstract

Purpose: To study the electrophysiological and pharmacological properties of the L-type Ca(2+) channel (LTCC) Ca(v)1.4alpha1 (alpha1F) subunit from mouse retina and assess their contributions to the native retinal channel.

Methods: The full-length cDNA of Ca(v)1.4alpha1 was cloned from murine retina in an RT-PCR approach. Ca(v)1.4alpha1 was expressed alone or together with the auxiliary alpha2delta1 and beta2a or beta3 subunits in HEK293 cells. The electrophysiological and pharmacological characteristics of L-type Ca(2+) and Ba(2+) inward currents (I(Ca) and I(Ba)) induced by Ca(v)1.4alpha1 were determined by the whole-cell configuration of the patch-clamp method and compared with currents induced by the cardiac and smooth muscle-type Ca(v)1.2alpha1 (alpha1C) channel.

Results: Ca(v)1.4alpha1-mediated I(Ba) was observed only when the alpha2delta1 and beta subunits were coexpressed. Current densities were approximately two times higher with beta2a than with beta3. I(Ba) activated faster and revealed much slower time-dependent inactivation than I(Ba) induced by Ca(v)1.2alpha1. Unlike in Ca(v)1.2alpha1, inactivation was not accelerated with Ca(2+) as the charge carrier, indicating the absence of Ca(2+)-dependent inactivation in Ca(v)1.4alpha1. Ca(v)1.4alpha1 exhibited voltage-dependent inactivation. The dihydropyridine (DHP) antagonist isradipine blocked Ca(v)1.4alpha1 with approximately 20-fold lower sensitivity than Ca(v)1.2alpha1. The agonistic DHP BayK 8644 stimulated maximum I(Ba) approximately sixfold. Ca(v)1.4alpha1 revealed only moderate sensitivities to L- and D-cis-diltiazem, with IC(50) in the micromolar range. Both enantiomers unexpectedly blocked Ca(v)1.4alpha1 with almost equal IC(50).

Conclusions: The data indicate that Ca(v)1.4alpha1 subunit constitutes the major molecular correlate of retinal L-type Ca(2+) current. Its intrinsic biophysical properties, in particular its unique inactivation properties, enable Ca(v)1.4alpha1 to provide a sustained I(Ca) over a voltage range such as required for tonic glutamate release at the photoreceptor synapse.