Mechanism of tetracaine block of cyclic nucleotide-gated channels.

Local anesthetics are a diverse group of ion channel blockers that can be used to probe conformational changes in the pore. We examined the effects of the local anesthetic tetracaine on rod and olfactory cyclic nucleotide-gated channels expressed from subunit 1 in Xenopus oocytes. We found that 40 microM tetracaine effectively blocked the bovine rod channel but not the rat olfactory channel at saturating concentrations of cGMP. By testing chimeric channels containing regions of sequence from both rod and olfactory channels, we found that determinants of apparent affinity for tetracaine at saturating cGMP did not map to any one region of the channel sequence. Rather, the differences in apparent affinity could be explained by differences between the chimeras in the free energy of the opening allosteric transition. If a channel construct (such as the rod channel) spent appreciable time in the closed state at saturating cGMP, then it had a high apparent affinity for tetracaine. If, on the other hand, a channel construct (such as the olfactory channel) spent little time in the closed state at saturating cGMP, then it had a low apparent affinity for tetracaine. Furthermore, tetracaine became more effective at low concentrations of cGMP and at saturating concentrations of cAMP, conditions which permit the channels to spend more time in the closed configuration. These results were well fit by a model in which tetracaine binds more tightly to the closed channel than to the open channel. Dose-response curves for tetracaine in the presence of saturating cGMP are well fit with a Michaelis-Menten binding scheme indicating that a single tetracaine molecule is sufficient to produce block. In addition, tetracaine block is voltage dependent with an effective z delta of +0.56. These data are consistent with a pore-block hypothesis. The finding that tetracaine is a state-dependent pore blocker suggests that the inner mouth of the pore of cyclic nucleotide-gated channels undergoes a conformational change during channel opening.