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Biophys. J. BioFAST: First Published April 8, 2005. doi:10.1529/biophysj.104.056804
© 2005 by the Biophysical Society.

A more recent version of this article appeared on June 1, 2005.
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CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING

Epithelial Na+ Channel Subunit Stoichiometry

Alexander Staruschenko 1*, Emily Adams 1, Rachell E Booth 2 and James D Stockand 1

1 University of Texas Health Science Center at San Antonio
2 Texas State University

* To whom correspondence should be addressed. E-mail: starushchenk{at}uthscsa.edu.

Submitted on November 22, 2004
Revised on December 8, 2004
Accepted on 29 March 2005


  Abstract
Ion channels, including the epithelial Na+ channel (ENaC), are intrinsic membrane proteins comprised of component subunits. Proper subunit assembly and stoichiometry are essential for normal physiological function of the channel protein. ENaC is comprised of three subunits, {alpha}, {beta} and {gamma}, that have common tertiary structures and much amino acid sequence identity. For maximal ENaC activity, each subunit is required. The subunit stoichiometry of functional ENaC within the membrane remains uncertain. We combined a biophysical approach, fluorescence intensity ratio (FIR) analysis, used to assess relative subunit stoichiometry with total internal reflection fluorescence (TIRF) microscopy, which enables isolation of plasma membrane fluorescence signals, to determine the limiting subunit stoichiometry of ENaC within the plasma membrane. Our results demonstrate that membrane ENaC contains equal numbers of each type of subunit and that at steady-state, subunit stoichiometry is fixed. Moreover, we find that when all three ENaC subunits are co-expressed, heteromeric channel formation is favored over homomeric channels. Electrophysiological results testing effects of ENaC subunit dose on channel activity were consistent with TIRF-FIR findings and confirmed preferential formation of heteromeric channels containing equal numbers of each subunit.

Key Words: Fluorescence resonance energy transfer, aldosterone, fluorescence intensity ratio, hypertension, total internal reflection fluorescence




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Copyright © 2005 by the Biophysical Society.