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Gating Transitions in Bacterial Ion Channels Measured at 3 µs Resolution

Shapalov, George and Lester, Henry A. (2004) Gating Transitions in Bacterial Ion Channels Measured at 3 µs Resolution. Journal of General Physiology, 124 (2). pp. 151-161. ISSN 0022-1295. http://resolver.caltech.edu/CaltechAUTHORS:SHAjgp04

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Abstract

Ion channels of high conductance (>200 pS) are widespread among prokaryotes and eukaryotes. Two examples, the Escherichia coli mechanosensitive ion channels Ec-MscS and Ec-MscL, pass currents of 125–300 pA. To resolve temporal details of conductance transitions, a patch-clamp setup was optimized for low-noise recordings at a time resolution of 3 µs (10–20 times faster than usual). Analyses of the high-resolution recordings confirm that Ec-MscL visits many subconductance states and show that most of the intersubstate transitions occur more slowly than the effective resolution of 3 µs. There is a clear trend toward longer transition times for the larger transitions. In Ec-MscS recordings, the majority of the observed full conductance transitions are also composite. We detected a short-lived (~20 µs) Ec-MscS substate at 2/3 of full conductance; transitions between 2/3 and full conductance did not show fine structure and had a time course limited by the achieved resolution. Opening and closing transitions in MscS are symmetrical and are not preceded or followed by smaller, rapid currents ("anticipations" or "regrets"). Compared with other, lower-conductance channels, these measurements may detect unusually early states in the transitions from fully closed to fully open. Increased temporal resolution at the single-molecule level reveals that some elementary steps of structural transitions are composite and follow several alternative pathways, while others still escape resolution. High-bandwidth, low-noise single-channel measurements may provide details about state transitions in other high-conductance channels; and similar procedures may also be applied to channel- and nanopore-based single-molecule DNA measurements.


Item Type:Article
Additional Information:© 2004 The Rockefeller University Press. RUP grants the public the non-exclusive right to copy, distribute, or display the Work under a Creative Commons Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/ and http://creativecommons.org/licenses/by-nc-sa/3.0/legalcode. Submitted: 3 May 2004. Accepted: 23 June 2004. Published online Jul 26 2004. doi:10.1085/jgp.200409087 We thank Fred Sigworth, Dennis Dougherty, and Doug Rees for guidance, Josef Dudel for instruction on optimizing headstages and quartz pipettes, Randal Bass and Yan Poon for protein samples, Lori Lee for providing unpublished data, Alan Finkel, Richard Lobdill, and Eric Fung for help with Axopatch and Digidata modifications, and Daniel Clayton for help with the manuscript and discussions. This work was supported by a grant from the National Institutes of Health (GM-062532).
Funders:
Funding AgencyGrant Number
National Institutes of HealthGM-062532
Subject Keywords:MscL; MscS; patch clamping; single channel; substate
Record Number:CaltechAUTHORS:SHAjgp04
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:SHAjgp04
Alternative URL:http://dx.doi.org/10.1085/jgp.200409087
Usage Policy:RUP grants the public the non-exclusive right to copy, distribute, or display the Work under a Creative Commons Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/ and http://creativecommons.org/licenses/by-nc-sa/3.0/legalcode.
ID Code:6313
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:01 Dec 2006
Last Modified:26 Dec 2012 09:20

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