Nuclear Membrane Electrophysiology of Ryanodine Receptor And Its Regulation by cADPR

Mobilization of intracellular calcium (Ca2+) through the ryanodine receptor (RyR) channels in the endoplasmic reticulum (ER) generates cellular signals that control virtually all aspects of life. The characterization of single RyR channel properties is therefore the fundamental to understand the generation and modulation of intracellular Ca2+ signaling events. Although the intracellular localization of RyR restricted its accessibility by conventional patch-clamp electrophysiology, single channel properties of RyR have been extensively studied by incorporating the channel proteins into artificial planar lipid bilayer. Up until now, the physiological properties of RyR channels in the native ER membrane have never been investigated. All the gating properties and ligand regulations of RyR single channel were obtained exclusively from the bilayer method. The single RyR channel characteristics and its regulations observed in the artificial membrane might not fully reflect the physiological behavior of the channel in the native membrane. To overcome this obstacle, we applied the nuclear membrane electrophysiology on a stable cell line expressing recombinant RyR channels which allows us to investigate single channel properties of RyR in the native membrane environment. We successfully recorded single channel activities on the nuclear membrane of the RyR expressing cells. The detected channel had all the characteristics of RyR, including the sensitivity to caffeine and cADPR and inhibition by ruthenium red. Our preliminary results demonstrated for the first time that single RyR channel activities can be studied in the native membrane. Furthermore, we identified a short sequence in the N-terminus of RyR protein which is conserved with the NAD binding pocket of the ADP-ribosyl cyclase. This homology sequence suggests that cADPR may activate RyR channel by direct binding to the receptor. In this research project, we will define the properties of single RyR channel, including the permeation properties and ligand regulations, in the native nuclear membrane and elucidate the activation of the RyR by cADPR using the nuclear membrane electrophysiology.
This proposal focuses on:
Aim 1. To characterize the permeation properties of single RyR channel in nuclear envelope. Aim 2. To characterize the ligand regulations of RyR channel in nuclear envelope.
Aim 3. To characterize the activation of RyR single channel by cADPR.