INTERACTIONS BETWEEN H-TYPE CALCIUM CHANNELS - EVIDENCE FOR INDEPENDENT CHANNEL-ASSOCIATED BINDING-SITES( AND CA2+ NEAR CARDIAC L)

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