Ca²⁺ is an important intracellular signal that regulates many cellular processes. Ryanodine receptors (RyR) mediate Ca²⁺ release from SR/ER and play important roles in intracellular Ca²⁺ signaling. The ability of the RyR channel to mobilize intracellular Ca ²⁺ depends on both its permeation and gating properties. Until now, the existence of Ca²⁺ flux passing through an open RyR pore to feed back and modulate gating of the channel is still uncertain, and the knowledge of permeation properties of RyR is inadequate.

The overall objective of this dissertation is to define some basic properties of RyR such as their Ca²⁺ regulation in physiological conditions, how (or if) a Ca²⁺ flux passing through an open RyR pore feeds back and modulates gating of the channel as well as the potential functional consequences of the channel's selectivity/permeability. The hypotheses of the project are: (1) Single RyR2 channels are modulated by Ca²⁺ feeding though their own pore. (2) Pharmacological agents that can alter the permeation of ryanodine receptor do so by changing the RyR pore to one of two conformations with different ion selectivity.

The RyR channels were reconstituted in planar lipid bilayers and channel regulation. The first part defines the Ca²⁺ sensitivity of single type-2 RyR channels under quasi-physiological condition. With cytosolic Mg²⁺ and ATP present, RyR activity was significantly reduced at all steady state cytosolic Ca²⁺ concentrations tested. The second part defines flux dependent regulation. Single RyR2 channel studies were done using 3 different cations (Ca²⁺, Mg²⁺, & Cs⁺ as control) as the ion fluxed through the pore. Ca ²⁺ and Mg²⁺ fluxes exhibit different regulatory patterns depending on flux amplitude. These flux dependent regulatory effects were abolished or reduced by addition of a fast cytosolic buffer. In the third part, different pharmacological agents were used to modify its conductance and selectivity to better understand the permeation properties of RyR2 pore. The pharmacological agents modified both RyR2 conductance and selectivity with different mechanisms. All of these results suggest that RyR calcium regulation is a complex process at both luminal and cytosolic sites.