The opening of Ca2+ release channels/ryanodine receptors (RyR) in cardiac sarcoplasmic reticulum (SR) is triggered by a rapid and transient influx of external Ca2+. Yet, previous studies aimed at elucidating the effect of modulators of RyRs have been conducted at a constant bath [Ca2+]. Since the response of RyRs depends on the rate of [Ca2+] application, the effect of putative modulators of Ca2+ release in a system where [Ca2+] raises and declines transitory fast, such as occurs in vivo during muscle contraction, is unknown. This proposal will study the kinetic response of RyRs to fast, calibrated, and transient elevations of myoplasmic [Ca2+]. Studies will be focused on investigating how the phosphorylation state of the RyRs affect their dynamic response to Ca2+. Native or protein kinase-treated RyRs will be fused to lipid bilayers and the [Ca2+] in the microenvironment of the channel will be elevated by flash photolysis of caged Ca2+. The protein kinases to be tested (Ca2+/calmodulin-dependent protein kinase, protein kinase A and protein kinase C) have been shown by several investigators, including the applicant and the sponsor, to regulate RyRs at constant bath [Ca2+]. Thus, they will likely affect the dynamic response of RyRs to [Ca2+] as well. This novel approach, which promises important and exciting data concerning modulation of RyRs by phosphorylation, will significantly broaden the scientific skills of the applicant and prepare him to carry out integral studies of RyRs.
