Local anesthetics are a diverse group of clinically useful compounds that act as pore blockers of both voltage- and cyclic nucleotide–gated (CNG) ion channels. We used the local anesthetic tetracaine to probe the nature of the conformational change that occurs in the pore of CNG channels during the opening allosteric transition. When applied to the intracellular side of wild-type rod CNG channels expressed in Xenopus oocytes from the α subunit, the local anesthetic tetracaine exhibits state-dependent block, binding with much higher affinity to closed states than to open states. Here we show that neutralization of a glutamic acid in the conserved P region (E363G) eliminated this state dependence of tetracaine block. Tetracaine blocked E363G channels with the same effectiveness at high concentrations of cGMP, when the channel spent 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 allosteric transition. These results suggest that E363 is accessible to internal tetracaine in the closed but not the open configuration of the pore and that the conformational change that accompanies channel opening includes a change in the conformation or accessibility of E363.
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1 November 1997
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November 01 1997
Tetracaine Reports a Conformational Change in the Pore of Cyclic Nucleotide–gated Channels
Anthony A. Fodor,
Anthony A. Fodor
From the Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, Washington 98195-7290
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Kevin D. Black,
Kevin D. Black
From the Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, Washington 98195-7290
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William N. Zagotta
William N. Zagotta
From the Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, Washington 98195-7290
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Anthony A. Fodor
From the Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, Washington 98195-7290
Kevin D. Black
From the Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, Washington 98195-7290
William N. Zagotta
From the Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, Washington 98195-7290
Address correspondence to William N. Zagotta, Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington School of Medicine, Box 357290, Seattle, WA 98195-7290. Fax: 206-543-0934; E-mail: [email protected]
Received:
June 06 1997
Accepted:
August 11 1997
Online ISSN: 1540-7748
Print ISSN: 0022-1295
1997
J Gen Physiol (1997) 110 (5): 591–600.
Article history
Received:
June 06 1997
Accepted:
August 11 1997
Citation
Anthony A. Fodor, Kevin D. Black, William N. Zagotta; Tetracaine Reports a Conformational Change in the Pore of Cyclic Nucleotide–gated Channels . J Gen Physiol 1 November 1997; 110 (5): 591–600. doi: https://doi.org/10.1085/jgp.110.5.591
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