Sarcoplasmic reticulum lumenal Ca2+ has access to cytosolic activation and inactivation sites of skeletal muscle Ca2+ release channel

The effects of sarcoplasmic reticulum lumenal (trans) Ca2+ on cytosolic (cis) ATP-activated rabbit skeletal muscle Ca2+ release channels (ryanodine receptors) were examined using the planar lipid bilayer method, Single channels were recorded in symmetric 0.25 M KCI media with K+ as the major current carrier. With nanomolar [Ca2+] in both bilayer chambers, the addition of 2 mM cytosolic ATP greatly increased the number of short channel openings. As lumenal [Ca2+] was increased from <0.1 mu M to similar to 250 mu M, increasing channel activities and events with long open time constants were seen at negative holding potentials. Channel activity remained low at positive holding potentials. Further increase in lumenal [Ca2+] to 1, 5, and 10 mM resulted in a decrease in channel activities at negative holding potentials and increased activities at positive holding potentials. A voltage-dependent activation by 50 mu M lumenal Ca(2+)was also observed when the channel was minimally activated by <1 mu M cytosolic Ca2+ in the absence of ATP. With mu M cytosolic Ca2+ in the presence or absence of 2 mM ATP, single-channel activities showed no or only a weak voltage dependence. Other divalent cations (Mg2+, Ba2+) could not replace lumenal Ca2+. On the contrary, cytosolic ATP-activated channel activities were decreased as lumenal Ca2+ fluxes were reduced by the addition of 1-5 mM BaCl2, or MgCl2, to the lumenal side, which contained 50 mu M Ca2+. An increase in [KCl] from 0.25 M to 1 M also reduced single-channel activities. Addition of the ''fast'' Ca2+ buffer 1,2-bis(2-aminophenoxy)ethaneletraacetic acid (BAPTA) to the cis chamber increased cytosolic ATP-, lumenal Ca2+-activated channel activities to a nearly maximum level. These results suggested that lumenal Ca2+ flowing through the skeletal muscle Ca2+ release channel may regulate channel activity by having access to cytosolic Ca2+ activation and Ca2+ inactivation sites that are located in ''BAPTA-inaccessible'' and ''BAPTA-accessible'' spaces, respectively.