Evidence for negative charge in the conduction pathway of the cardiac ryanodine receptor channel provided by the interaction of K+ channel N-type inactivation peptides

We have investigated the interaction of two peptides (ShB - net charge +3 and ShB:E12KDI3K - net charge +7) derived from the NH2-terminal domain of the Shaker K+ channel with purified, ryanodine-modified, cardiac Ca2+-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a single-site binding scheme with 50% saturation occurring at 5.92 +/- 1.06 mu M for ShB and 0.59 +/- 0.14 nM for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 +/- 0.4 sec(-1) mu M-1 for ShB and 2000 +/- 200 sec(-1) mu M-1 for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied potential with the bulk of the voltage-dependence residing in K-off. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As is the case with inactivation and block of K+ channels, this is mediated by a large increase in K-on. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites which will contribute to the ion handling properties of this channel.