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Title: The tarantula toxin GxTx detains K + channel gating charges in their resting conformation

Abstract

Allosteric ligands modulate protein activity by altering the energy landscape of conformational space in ligand–protein complexes. Here we investigate how ligand binding to a K + channel’s voltage sensor allosterically modulates opening of its K + -conductive pore. The tarantula venom peptide guangxitoxin-1E (GxTx) binds to the voltage sensors of the rat voltage-gated K + (Kv) channel Kv2.1 and acts as a partial inverse agonist. When bound to GxTx, Kv2.1 activates more slowly, deactivates more rapidly, and requires more positive voltage to reach the same K + -conductance as the unbound channel. Further, activation kinetics are more sigmoidal, indicating that multiple conformational changes coupled to opening are modulated. Single-channel current amplitudes reveal that each channel opens to full conductance when GxTx is bound. Inhibition of Kv2.1 channels by GxTx results from decreased open probability due to increased occurrence of long-lived closed states; the time constant of the final pore opening step itself is not impacted by GxTx. When intracellular potential is less than 0 mV, GxTx traps the gating charges on Kv2.1’s voltage sensors in their most intracellular position. Gating charges translocate at positive voltages, however, indicating that GxTx stabilizes the most intracellular conformation of the voltage sensors (their restingmore » conformation). Kinetic modeling suggests a modulatory mechanism: GxTx reduces the probability of voltage sensors activating, giving the pore opening step less frequent opportunities to occur. This mechanism results in K + -conductance activation kinetics that are voltage-dependent, even if pore opening (the rate-limiting step) has no inherent voltage dependence. We conclude that GxTx stabilizes voltage sensors in a resting conformation, and inhibits K + currents by limiting opportunities for the channel pore to open, but has little, if any, direct effect on the microscopic kinetics of pore opening. The impact of GxTx on channel gating suggests that Kv2.1’s pore opening step does not involve movement of its voltage sensors.« less

Authors:
ORCiD logo [1]; ORCiD logo [2];  [3];  [2]; ORCiD logo [2]; ORCiD logo [2]; ORCiD logo [4]
  1. Department of Physiology & Membrane Biology, University of California, Davis, Davis, CA
  2. Neurobiology Course, Marine Biological Laboratory, Woods Hole, MA
  3. Department of Physiology & Membrane Biology, University of California, Davis, Davis, CA, Neurobiology Course, Marine Biological Laboratory, Woods Hole, MA
  4. Department of Physiology & Membrane Biology, University of California, Davis, Davis, CA, Neurobiology Course, Marine Biological Laboratory, Woods Hole, MA, Department of Anesthesiology and Pain Medicine, University of California, Davis, Davis, CA
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1480880
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Journal Article: Published Article
Journal Name:
Journal of General Physiology
Additional Journal Information:
Journal Name: Journal of General Physiology Journal Volume: 151 Journal Issue: 3; Journal ID: ISSN 0022-1295
Publisher:
Rockefeller University Press
Country of Publication:
United States
Language:
English

Citation Formats

Tilley, Drew C., Angueyra, Juan M., Eum, Kenneth S., Kim, Heesoo, Chao, Luke H., Peng, Anthony W., and Sack, Jon T.. The tarantula toxin GxTx detains K + channel gating charges in their resting conformation. United States: N. p., 2018. Web. doi:10.1085/jgp.201812213.
Tilley, Drew C., Angueyra, Juan M., Eum, Kenneth S., Kim, Heesoo, Chao, Luke H., Peng, Anthony W., & Sack, Jon T.. The tarantula toxin GxTx detains K + channel gating charges in their resting conformation. United States. doi:10.1085/jgp.201812213.
Tilley, Drew C., Angueyra, Juan M., Eum, Kenneth S., Kim, Heesoo, Chao, Luke H., Peng, Anthony W., and Sack, Jon T.. Mon . "The tarantula toxin GxTx detains K + channel gating charges in their resting conformation". United States. doi:10.1085/jgp.201812213.
@article{osti_1480880,
title = {The tarantula toxin GxTx detains K + channel gating charges in their resting conformation},
author = {Tilley, Drew C. and Angueyra, Juan M. and Eum, Kenneth S. and Kim, Heesoo and Chao, Luke H. and Peng, Anthony W. and Sack, Jon T.},
abstractNote = {Allosteric ligands modulate protein activity by altering the energy landscape of conformational space in ligand–protein complexes. Here we investigate how ligand binding to a K + channel’s voltage sensor allosterically modulates opening of its K + -conductive pore. The tarantula venom peptide guangxitoxin-1E (GxTx) binds to the voltage sensors of the rat voltage-gated K + (Kv) channel Kv2.1 and acts as a partial inverse agonist. When bound to GxTx, Kv2.1 activates more slowly, deactivates more rapidly, and requires more positive voltage to reach the same K + -conductance as the unbound channel. Further, activation kinetics are more sigmoidal, indicating that multiple conformational changes coupled to opening are modulated. Single-channel current amplitudes reveal that each channel opens to full conductance when GxTx is bound. Inhibition of Kv2.1 channels by GxTx results from decreased open probability due to increased occurrence of long-lived closed states; the time constant of the final pore opening step itself is not impacted by GxTx. When intracellular potential is less than 0 mV, GxTx traps the gating charges on Kv2.1’s voltage sensors in their most intracellular position. Gating charges translocate at positive voltages, however, indicating that GxTx stabilizes the most intracellular conformation of the voltage sensors (their resting conformation). Kinetic modeling suggests a modulatory mechanism: GxTx reduces the probability of voltage sensors activating, giving the pore opening step less frequent opportunities to occur. This mechanism results in K + -conductance activation kinetics that are voltage-dependent, even if pore opening (the rate-limiting step) has no inherent voltage dependence. We conclude that GxTx stabilizes voltage sensors in a resting conformation, and inhibits K + currents by limiting opportunities for the channel pore to open, but has little, if any, direct effect on the microscopic kinetics of pore opening. The impact of GxTx on channel gating suggests that Kv2.1’s pore opening step does not involve movement of its voltage sensors.},
doi = {10.1085/jgp.201812213},
journal = {Journal of General Physiology},
issn = {0022-1295},
number = 3,
volume = 151,
place = {United States},
year = {2018},
month = {11}
}

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