CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING |
Stoichiometric pore mutations of the GABAAR reveal a pattern of hydrogen bonding with picrotoxin
Brian E Erkkila 1, Anna V Sedelnikova 1 and David S. Weiss 2*
1 UTHSCSA
2 University of Texas Health Science Center at San Antonio
* To whom correspondence should be addressed. E-mail: weissd{at}uthscsa.edu.
Submitted on September 20, 2007
Revised on October 22, 2007
Accepted on 22 January 2008
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Abstract |
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Picrotoxin (PTX) is a non-competitive antagonist of many ligand-gated ion channels, with a site of action believed to be within the ion-conducting pore. In the GABAA receptor, a threonine residue in the second transmembrane domain (M2) is of particular importance for the binding of, and ultimate inhibition by, PTX. To better understand the relationship between this residue and the PTX molecule, we mutated this threonine residue to serine, valine and tyrosine to change the structural and biochemical characteristics at this location. The known subunit stoichiometry of the GABAAR allowed us to create receptors with anywhere from zero to five mutations. With an increasing number of mutated subunits, each amino acid substitution revealed a unique pattern of changes in PTX sensitivity, ultimately encompassing sensitivity shifts over several orders of magnitude. The electrophysiological data on PTX-mediated block, and supporting modeling and docking studies, provide evidence that an interaction between the PTX molecule and three adjacent uncharged polar amino acids at this position of the pore are crucial for PTX-mediated inhibition.
Key Words:
antagonist, ligand-gated ion channel, mutagenesis, receptor, structure/function
