Interactions between H+ and Ca2+ near cardiac L-type calcium channels: evidence for independent channel-associated binding sites.

Monovalent and divalent ions are known to affect voltage-gated ion channels by the screening of, and/or binding to, negative charges located on the surface of cell membranes within the vicinity of the channel protein. In this investigation, we studied gating shifts of cardiac L-type calcium channels induced by extracellular H+ and Ca2+ to determine whether these cations interact at independent or competitive binding sites. At constant pHo (7.4), Cao-induced gating shifts begin to approach a maximum value (approximately equal to 17 mV) at concentrations of extracellular calcium of > or = 40 mM. A fraction of the calcium-dependent gating shift could be titrated with an effective pKa = 6.9 indicating common and competitive access to H+ and Ca2+ ions for at least one binding site. However, if pHo is lowered when Cao is > or = 40 mM, additional shifts in gating are measured, suggesting a subpopulation of sites to which Ca2+ and H+ bind independently. The interdependence of L-channel gating shifts and Cao and pHo was well described by the predictions of surface potential theory in which two sets of binding sites are postulated; site 1 (pKa = 5.5) is accessible only to H+ ions and site 2 (pKa = 6.9) is accessible to both Ca2+ and H+ ions. Theoretical computations generated with this model are consistent with previously determined data, in which interactions between these two cations were not studied, in addition to the present experiments in which interactions were systematically probed.