Most voltage-gated K+ currents are relatively insensitive to extracellular Na+ (Na+o), but Na+o potently inhibits outward human ether-a-go-go–related gene (HERG)–encoded K+ channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429–30). We studied wild-type (WT) and mutant HERG currents and used two strategic probes, intracellular Na+ (Na+i) and extracellular Ba2+ (Ba2+o), to define a site where Na+o interacts with HERG. Currents were recorded from transfected Chinese hamster ovary (CHO-K1) cells using the whole-cell voltage clamp technique. Inhibition of WT HERG by Na+o was not strongly dependent on the voltage during activating pulses. Three point mutants in the P-loop region (S624A, S624T, S631A) with intact K+ selectivity and impaired inactivation each had reduced sensitivity to inhibition by Na+o. Quantitatively similar effects of Na+i to inhibit HERG current were seen in the WT and S624A channels. As S624A has impaired Na+o sensitivity, this result suggested that Na+o and Na+i act at different sites. Extracellular Ba2+ (Ba2+o) blocks K+ channel pores, and thereby serves as a useful probe of K+ channel structure. HERG channel inactivation promotes relief of Ba2+ block (Weerapura, M., S. Nattel, M. Courtemanche, D. Doern, N. Ethier, and T. Hebert. 2000. J. Physiol. 526:265–278). We used this feature of HERG inactivation to distinguish between simple allosteric and pore-occluding models of Na+o action. A remote allosteric model predicts that Na+o will speed relief of Ba2+o block by promoting inactivation. Instead, Na+o slowed Ba2+ egress and Ba2+ relieved Na+o inhibition, consistent with Na+o binding to an outer pore site. The apparent affinities of the outer pore for Na+o and K+o as measured by slowing of Ba2+ egress were compatible with competition between the two ions for the channel pore in their physiological concentration ranges. We also examined the role of the HERG closed state in Na+o inhibition. Na+o inhibition was inversely related to pulsing frequency in the WT channel, but not in the pore mutant S624A.
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1 October 2002
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September 16 2002
Extracellular Sodium Interacts with the HERG Channel at an Outer Pore Site
Franklin M. Mullins,
Franklin M. Mullins
1Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232
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Svetlana Z. Stepanovic,
Svetlana Z. Stepanovic
2Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, TN 37232
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Reshma R. Desai,
Reshma R. Desai
1Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232
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Alfred L. George, Jr.,
Alfred L. George, Jr.
1Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232
3Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232
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Jeffrey R. Balser
Jeffrey R. Balser
1Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232
2Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, TN 37232
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Franklin M. Mullins
1Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232
Svetlana Z. Stepanovic
2Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, TN 37232
Reshma R. Desai
1Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232
Alfred L. George, Jr.
1Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232
3Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232
Jeffrey R. Balser
1Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232
2Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, TN 37232
Address correspondence to Jeffrey R. Balser, Room 560 PRB (MRB II), Vanderbilt University School of Medicine, Nashville, TN 37232. Fax: (615) 936-0456; E-mail: [email protected]
Portions of this work were previously published in abstract form (Mullins, F.M., R.R. Desai, A.L. George, Jr., and J.R. Balser. 2001. Biophys. J. 80:216A; and Mullins, F.M., S.Z. Stepanovic, and J.R. Balser. 2002. Biophys. J. 82:579a).
*
Abbreviations used in this paper: CHO, chinese hamster ovary; HERG, human ether-a-go-go–related gene.
Received:
March 05 2002
Revision Received:
July 15 2002
Accepted:
July 23 2002
Online ISSN: 1540-7748
Print ISSN: 0022-1295
The Rockefeller University Press
2002
J Gen Physiol (2002) 120 (4): 517–537.
Article history
Received:
March 05 2002
Revision Received:
July 15 2002
Accepted:
July 23 2002
Citation
Franklin M. Mullins, Svetlana Z. Stepanovic, Reshma R. Desai, Alfred L. George, Jeffrey R. Balser; Extracellular Sodium Interacts with the HERG Channel at an Outer Pore Site . J Gen Physiol 1 October 2002; 120 (4): 517–537. doi: https://doi.org/10.1085/jgp.20028589
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