Cross-Reactivity of Ryanodine Receptors with Putative Modulators of Ion Channels in the Plasma Membrane

Abstract

The role of specific ion channels for cell function is often investigated using “pharmacological agents” (PA). PA specificity is typically determined using the whole-cell voltage clamp technique, which allows for the contrasting of many plasma membrane ion channels but not intracellular calcium release channels such as the ryanodine receptors (RyRs), which are capable of triggering numerous cellular processes. We determined that an UV-visible cuvette assay can be utilized for the screening of RyR cross-reactivity with PA, allowing for rapid determination of modulators that affect the rate of Ca2+ leak from skeletal muscle sacroplasmic reticulum microsomes. Agents that tested positive (both agonists and antagonists) were also tested on skeletal RyRs reconstituted into planar lipid bilayers. One of our main focus was TRP modulators, as early evidence suggested that these agents could target RyRs. At doses normally used to modulate TRP channels, menthol activated and anandamide inhibited RyRs. On the contrary, pseudocapsaicin and capsaicin were without effect. We have hypothesized that the cytosolic vestibular regions in the RyRs conduction pathway could have structural homology with that of voltage-gated Na+ and K+ channels. We then tested agents thought to act on the cytosolic vestibular region of those channels and determined that lamotrigine isethionate (Na+ channel blockers) and UCL1964 (K+ channel blocker) also block RyRs. Thus, our results suggest that RyR channels are targets for various ion channel modulators and could mediate some of their therapeutic actions. Exploring RyRs cross-reactivity has the potential for identifying pharmacological tools to better understand RyRs gating and RyR-mediated Ca2+ signaling in cells. (Supported by NIH R01 GM078665 to JAC)