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 mu M 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 ap parent affinity for tetracaine at saturating cGMP did not map to any o ne region of the channel sequence. Rather, the differences in apparent affinity could be explained by differences between the chimeras in th e free energy of the opening allosteric transition. If a channel const ruct (such as the rod channel) spent appreciable time in the closed st ate at saturating cGMP, then it had a high apparent affinity for tetra caine. If, on the other hand, a channel construct (such as the olfacto ry channel) spent little time in the closed state at saturating cGMP, then it had a low apparent affinity for tetracaine. Furthermore, tetra caine became more effective at low concentrations of cGMP and at satur ating concentrations of cAMP, conditions which permit the channels to spend more time in the closed configuration. These results were well f it by a model in which tetracaine binds more tightly to the closed cha nnel than to the open channel. Dose-response curves for tetracaine in the presence of saturating cGMP are well fit with a Michaelis-Menten b inding scheme indicating that a single tetracaine molecule is sufficie nt to produce block. In addition, tetracaine block is voltage dependen t with an effective z delta of +0.56. These data are consistent with a pore-block hypothesis. The finding that tetracaine is a state-depende nt pore blocker suggests that the inner mouth of the pore of cyclic nu cleotide-gated channels undergoes a conformational change during chann el opening.