SARCOPLASMIC-RETICULUM LUMENAL CA2-MUSCLE CA2+ RELEASE CHANNEL( HAS ACCESS TO CYTOSOLIC ACTIVATION AND INACTIVATION SITES OF SKELETAL)

The effects of sarcoplasmic reticulum lumenal (trans) Ca2+ on cytosoli c (cis) ATP-activated rabbit skeletal muscle Ca2+ release channels (ry anodine receptors) were examined using the planar lipid bilayer method , Single channels were recorded in symmetric 0.25 M KCI media with Kas the major current carrier. With nanomolar [Ca2+] in both bilayer ch ambers, the addition of 2 mM cytosolic ATP greatly increased the numbe r 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 potential s. Channel activity remained low at positive holding potentials. Furth er increase in lumenal [Ca2+] to 1, 5, and 10 mM resulted in a decreas e in channel activities at negative holding potentials and increased a ctivities at positive holding potentials. A voltage-dependent activati on by 50 mu M lumenal Ca(2+)was also observed when the channel was min imally 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-ch annel activities showed no or only a weak voltage dependence. Other di valent cations (Mg2+, Ba2+) could not replace lumenal Ca2+. On the con trary, cytosolic ATP-activated channel activities were decreased as lu menal Ca2+ fluxes were reduced by the addition of 1-5 mM BaCl2, or MgC l2, 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. Addit ion of the ''fast'' Ca2+ buffer 1,2-bis(2-aminophenoxy)ethaneletraacet ic acid (BAPTA) to the cis chamber increased cytosolic ATP-, lumenal C a2+-activated channel activities to a nearly maximum level. These resu lts suggested that lumenal Ca2+ flowing through the skeletal muscle Ca 2+ release channel may regulate channel activity by having access to c ytosolic Ca2+ activation and Ca2+ inactivation sites that are located in ''BAPTA-inaccessible'' and ''BAPTA-accessible'' spaces, respectivel y.