Despite years of intense effort, the role of Ca+(2) in regulation of SR Ca+(2) release in skeletal muscle has not yet been clearly defined. The fundamental obstacle has been that all established functional assays of SR Ca+(2) release rely on Ca+(2) flux through the Ca+(2) channel. The result is that the free [Ca+(2)] in the microenvironment of the SR Ca+(2) release channel could not be precisely controlled. This problem is compounded in situ since Ca+(2) is thought to be released into a small morphologically- confined, diffusion-limited space (i.e., the T-SR cleft). In this proposal, the role of Ca+(2) in regulation (activation and inactivation) of SR Ca+(2) release in skeletal muscle is defined, for the first time, with the [Ca+(2)] in the T-SR cleft precisely controlled. The Ca+(2) control problem of past studies is overcome here by: 1) developing single channel and skinned fiber assays which rely on K+ or Cs+ (not Ca+(2)) flux (i.e., K+ and Cs+ do not regulate SR Ca+(2) channels); and 2) by using fast calibrated photo-manipulations of free [Ca+(2)] (via caged- Ca+(2) and caged Ca+(2)- chelator).
The specific aims are: 1) to specify how much Ca+(2) is """"""""actually"""""""" required to activate SR Ca+(2) release in situ, 2) to determine if Ca+(2) activates SR Ca+(2) release in response to T-tubule depolarization, 3) to define the underlying molecular basis of """"""""inactivation"""""""" of SR Ca+(2) release, 4) to determine if """"""""inactivation"""""""" in situ is mediated by direct binding of Ca+(2) to a site on the SR Ca+(2) channel, and 5) to determine the distances from SR membrane of the Ca+(2)- activation site, the Ca+(2)-inactivation site, and the channel mouths on the SR Ca+(2) channel protein. Defining how Ca+(2) regulates SR Ca+(2) release is important since SR Ca+(2) release represents a step at which muscle contractility can be modulated. It is clinically significant, therefore, as a potential point for pathological failure and therapeutic intervention in skeletal muscle. Further, defining how Ca+(2) regulates SR Ca+(2) release channels in skeletal muscle has broader significance since the regulation of analogous channels in other tissues (ryanodine and IP(3) receptor channels in smooth muscle, cardiac muscle, brain, and liver) are not well understood.
