Tetracaine is a tertiary amine local anaesthetic which inhibits ryanodine receptors (RyRs), the calcium release channels of the sarcoplasmic reticulum (SR). Tetracaine has been extensively used to study the role of the SR Ca(2+) fluxes in muscle cells, yet a detailed understanding of tetracaine action on RyR channels is lacking. Here we investigate tetracaine effects in single channel recording of sheep cardiac RyRs in lipid bilayers. Tetracaine decreased channel conductance (block) and open probability (inhibition). The IC(50) for inhibition had complex dependencies on membrane voltage and cytoplasmic [ATP], [Ca(2+)] and pH. We identify three mechanisms underlying these actions. First, a voltage-dependent, slow inhibition in which luminal and cytoplasmic tetracaine compete for a common neutral/cation binding site within the trans-membrane RyR domain to induce long closed events (~100 ms). The apparent binding rate is proportional to the RyR closed probability, indicating that it only operates on closed channels. Second, a voltage-independent, pH sensitive fast inhibition in which cytoplasmic and luminal tetracaine compete for a site located on the cytoplasmic domain of the RyR to induce fast closed events (~2 ms). Its IC(50) is not dependent on the open/closed conformation of RyR. Finally, a voltage-dependent block of the channel by cytoplasmic tetracaine reduced channel conductance. We develop a model for tetracaine inhibition which predicts that under diastolic conditions, i.e. when RyRs are mainly closed, the slow mechanism has the highest potency (IC(50)~200 μM) of the three mechanisms and is therefore the dominant form of inhibition. However, during periods of Ca(2+) release, i.e. when RyRs are open, the slow mechanism becomes ineffective, leaving the fast inhibition (IC(50)~2 mM) as the dominant effect. Because of this closed state inhibition property, tetracaine loses its efficacy when RyRs open. This has the effect of increasing the feedback on SR Ca(2+) release generated by cytoplasmic and luminal Ca(2+).
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