In cardiac myocytes, there are multiple Ca2+ entry pathways which may participate in triggering Ca2+ release from the sarcoplasmic reticulum (SR). Since the spatial distribution, kinetics of Ca2+ transport, and the micro-environment for various Ca2+ transporters are not the same, the efficacies and/or the mechanisms of these Ca2+ entry pathways in activating Ca2+ release can be very different. Our objective is to investigate the cellular mechanisms of controlling Ca2+ release by L-type Ca2+ channel, T-type Ca2+ channel, Na+-Ca2+ exchanger, and Na+ channel. Based on previous studies and our preliminary data, we proposed the following hypotheses. First, the characteristics Ca2+ release induced by Ca2+ current (I[ca]) is determined by the functional coupling of L-type Ca2+ channels and Ca2+ release channels (ryanodine receptors) in the junctional SR, and the uncoupling of these channels in the corbular SR. Second, Na+-Ca2+ exchanger, T-type Ca2+ channels, and Na+ channel activate Ca2+ release via mechanisms/pathways different from I[ca]-induced Ca2+ release, and their efficacies varies among animal species depend on the availability, distribution, and activity of the ion channel or transporter. To test the first hypothesis, we will (1) determine whether Ca2+ channels and ryanodine receptors are functionally coupled, and if so, whether such coupling is essential for the I[Ca]-induced fast Ca2+ release, and (2) determine whether I[Ca] can activate regenerative (partial or complete) Ca2+ release, and if so, whether such regenerative Ca2+ release involves corbular SR. To test the second hypothesis, we will (1) determine whether the Na+-Ca2+ exchanger mediated Ca2+ release is similar to the I[ca]-induced-Ca2+ release or the Ca2+-overload induced spontaneous oscillation, (2) examine the Na+-Ca2+ exchange mediated Ca2+ release and the possible Ca2+ release activated indirectly by Na+ influx and accumulation in animal species with higher exchanger activities, and (3) determine whether the transient Ca2+ entry via the T-type Ca2+ channel activates Ca2+ release, and if so, what is the difference between this T- type Ca2+ channel activated Ca2+ release and those activated by L-type Ca2+ channel and Na+-Ca2+ exchanger. In the proposed experiments, a combination of patch-clamp and Indo-1 fluorescence techniques will be applied to single isolated ventricular myocytes to monitor simultaneously the trans-membrane currents, and intracellular Ca2+ concentration. This project will provide important information on the control mechanisms of cardiac excitation-contraction coupling, and improve our understanding on Ca2+ signalling and processing in cardiac myocytes.
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