Abstract
We have investigated the molecular mechanisms whereby the I-II loop controls voltage-dependent inactivation in P/Q calcium channels. We demonstrate that the I-II loop is localized in a central position to control calcium channel activity through the interaction with several cytoplasmic sequences; including the III-IV loop. Several experiments reveal the crucial role of the interaction between the I-II loop and the III-IV loop in channel inactivation. First, point mutations of two amino acid residues of the I-II loop of Ca(v)2.1 (Arg-387 or Glu-388) facilitate voltage-dependent inactivation. Second, overexpression of the III-IV loop, or injection of a peptide derived from this loop, produces a similar inactivation behavior than the mutated channels. Third, the III-IV peptide has no effect on channels mutated in the I-II loop. Thus, both point mutations and overexpression of the III-IV loop appear to act similarly on inactivation, by competing off the native interaction between the I-II and the III-IV loops of Ca(v)2.1. As they are known to affect inactivation, we also analyzed the effects of beta subunits on these interactions. In experiments in which the beta(4) subunit is co-expressed, the III-IV peptide is no longer able to regulate channel inactivation. We conclude that (i) the contribution of the I-II loop to inactivation is partly mediated by an interaction with the III-IV loop and (ii) the beta subunits partially control inactivation by modifying this interaction. These data provide novel insights into the mechanisms whereby the beta subunit, the I-II loop, and the III-IV loop altogether can contribute to regulate inactivation in high voltage-activated calcium channels.
Highlights
We have investigated the molecular mechanisms whereby the I-II loop controls voltage-dependent inactivation in P/Q calcium channels
We demonstrate that the I-II loop is localized in a central position to control calcium channel activity through the interaction with several cytoplasmic sequences; including the III-IV loop
 subunits appear to affect the rate of L-type channel inactivation, steady-state inactivation seems to be less dependent on  subunit coexpression than non-L-type channels [9, 18, 23,24]
Summary
We have investigated the molecular mechanisms whereby the I-II loop controls voltage-dependent inactivation in P/Q calcium channels. In the midst of this apparent complexity, and contrary to all expectations, it was found that voltage and calcium may use the same molecular determinants to inactivate calcium channels [9] This basic observation points to the possibility that there may be an elusive mechanism whereby the various structural elements identified so far are coordinated to produce inactivation in calcium channels. A further challenge in solving the issue of calcium channel inactivation stems from a still greater level of complexity that is introduced by  subunit regulation These auxiliary subunits possess an enormous potential for in vivo tuning of channel behavior [2, 10]. The most interesting candidate sequence to investigate in terms of -dependent inactivation
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