Structure-function relationships of the ryanodine receptor

Abstract

Ryanodine receptors form a family of Ca2+ activated Ca2+ release channels localized to the membrane of the endo/sarcoplasmic reticulum. During excitationcontraction coupling in muscle, depolarization of the plasma membrane activates dihydropvridine receptors (DHPRs), which in turn trigger ryanodine receptors to release the Ca2+ necessary to elicit a contraction. In cardiac muscle, Ca2+ influx via the cardiac DHPR is thought to activate the cardiac ryanodine receptor, RyR2, while in contrast, Ca2+ influx in skeletal muscle is not required to activate the skeletal isoform, RyRl. However, it is possible that the skeletal DHPR allosterically causes an increased Ca2+ sensitivity of RyR l such that resting levels of cytoplasmic Ca2+ are sufficient to activate RyRl. In the first part of my dissertation. I used whole-cell patch clamp and photometric measurement of cytoplasmic Ca2+ in dyspedic myotubes expressing E4032A-RyRl to examine the role of Ca2+ activation in skeletal-type excitation-contraction coupling, because the point mutation, E4032A, was shown in lipid bilayers to drastically reduce the Ca2+ activation of RyR l. I found that Ca2+ activation is not required for the initiation of excitation-contraction coupling. Because amino acids 720-765 of the DHPR II-III loop and 1837-2168 (sR16) of RyRl were shown to physically interact in yeast two-hybrid assays, I studied, in the second part of my dissertation, both a chimera in which sR16 was substituted into RyR2 and that chimera’s inverse to test the functional importance of this interaction. My results suggest that sR16 participates in, but is not necessary for, skeletal-type excitation-contraction coupling. In the third part of my dissertation, I studied four cysteines in the RyRl carboxy-terminal domain that are conserved among all isoforms of ryanodine receptors and inositol 1,4,5-triphosphate receptors, a second family of Ca2+ release channels. When I tested mutation to serine of these four cysteines (C4876S, C4882S, C4958S, C4961S) in RyRl, I found that Ca2+ release function was eliminated. However, DHPR Ca2+ current enhancement was partially maintained suggesting that tetramerization of RyRl was unaffected since the receptor could functionally interact with the DHPR.