S-Adenosyl-L-Methionine Induced RyR2 Subconductance: Evidence for an Allosteric Mechanism

Abstract

We previously reported (Kampfer & Balog Biochem 49:7600, 2010) that S-adenosyl-l-methionine (SAM) can act as a RyR2 channel regulatory ligand in a manner independent from its recognized role as a biological methyl group donor. Channel activation appeared to arise from the interaction of SAM with a RyR2 adenine nucleotide binding site. In addition to its ability to activate RyR2, single channel recordings revealed distinct effects of SAM on RyR2 conductance, which we explored here in greater detail. The effects of SAM on native RyR2 channel conductance in symmetric cesium methanesulfonate were dependent on SAM concentration and holding potential. At negative potentials, cis SAM induced a single, clearly resolved subconductance state (∼2/3 full conductance). The proportion of SAM induced subconductance openings, as a proportion of all openings (Psub/Po), increased with decreasing negative potential. Kinetic analysis revealed that changes in the SAM off rate accounted for the voltage dependence of the transitions between the full open and SAM induced subconductance state. In contrast, at positive potentials SAM caused a striking reduction in channel openings with no distinct effect on channel conductance. Inconsistent with a simple pore block mechanism was the finding that the prevalence of the subconductance state was unaffected by varying the cesium concentration gradient across the bilayer. Furthermore, ATP but not 4-chloro-m-cresol, interfered with the effects of SAM at both negative and positive potentials, suggesting ATP competition with SAM for a common binding site. We interpret these findings in light of an allosteric mechanism whereby SAM interacts with a RyR2 adenine nucleotide binding site. SAM binds to and stabilizes a channel conformation of reduced conductance and we propose the voltage dependence of the SAM-induced subconductance state arises from a voltage-driven alteration in the affinity of the SAM binding site.