TETRACAINE REPORTS A CONFORMATIONAL CHANGE IN THE PORE OF CYCLIC NUCLEOTIDE-GATED CHANNELS

Local anesthetics are a diverse group of clinically useful compounds t hat act as pore blockers of both voltage-and cyclic nucleotide-gated ( CNG) ion channels. We used the local anesthetic tetracaine to probe th e nature of the conformational change that occurs in the pore of CNG c hannels during the opening allosteric transition. When applied to the intracellular side of wild-type rod CNG channels expressed in Xenopus oocytes from the or subunit, the local anesthetic tetracaine exhibits state-dependent block, binding with much higher affinity to closed sta tes than to open states. Here we show that neutralization of a glutami c acid in the conserved P region (E363G) eliminated this state depende nce of tetracaine block. Tetracaine blocked E363G channels with the sa me effectiveness at high concentrations of cGMP, when the channel spen t more time open, and at low concentrations of cGMP, when the channel spent more time closed. In addition, Ni2+, which promotes the opening allosteric transition, decreased the effectiveness of tetracaine block of wild-type but not E363G channels. Similar results were obtained in a chimeric CNG channel that exhibits a more favorable opening alloste ric transition. These results suggest that E363 is accessible to inter nal tetracaine in the closed but not the open configuration of the por e and that the conformational change that accompanies channel opening includes a change in the conformation or accessibility of E363.