Cardiac Ryanodine Receptor Channel Gating is Modified during Blocker-Induced Changes in Ion Permeation

Abstract

Ion channel blockers such as large quaternary ammonium (QA) compounds have traditionally been used as effective tools for looking at the interplay between ion permeation, channel gating and block in various voltage/ligand-gated K+ channels. Although the availability of detailed structural information regarding the channel pore has facilitated the interpretation of data from these channels, the blocker-induced full conductance (complete) block of ionic flux makes it indistinguishable from the normal closed or inactivated events. Therefore, the transitions between the blocked and closed or inactivated states are not directly visible in the single channel data, leading to loss of vital information pertaining to channel gating behavior.The cardiac ryanodine receptor (RyR2) is a massive (∼2.2MDa) Ca2+- activated Ca2+ release channel responsible for transducing the information from an incoming action potential to trigger cardiac contraction. It is a cation selective channel that exhibits a large single channel conductance for K+ (∼723 pS in 210 mM K+) but is only blocked to subconductance states by QA blockers, allowing detailed study of state transitions in the presence of an ongoing block. In this study, the kinetics of single recombinant mouse RyR2 channels reconstituted in planar lipid bilayers were examined in the presence of the QA blockers tetrabutyl (TBA)- and tetrapentyl ammonium (TPeA). Detailed analyses of single channel data and subsequent modeling of gating behavior using Hidden Markov model based algorithms reveals novel effects of blockers which differentially affect gating in the putative selectivity filter and the inner helix bundle crossing regions of the RyR2 pore, and provides further evidence in favor of the possible existence of these two distinct gating entities in the absence of direct evidence from crystal structures. Research supported by British Heart Foundation.