CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING |
Mechanosensitive channel gating transitions resolved by functional changes upon pore modification
Jessica L Bartlett 1, Yuezhou Li 1 and Paul Blount 1*
1 University of Texas Southwestern Medical Center
* To whom correspondence should be addressed. E-mail: paul.blount{at}utsouthwestern.edu.
Submitted on April 27, 2006
Revised on June 19, 2006
Accepted on 15 August 2006
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Abstract |
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The Mechanosensitive Channel of Large conductance (MscL) acts as a biological 'emergency release valve' that protects bacterial cells from hypoosmotic stress. While structural and functional studies and molecular dynamic simulations of this channel have led to several models for the structural transitions that occur in the gating process, inconsistencies linger and details are lacking. A previous study, using a method coined as the 'in vivo SCAM', identified several residues in the channel pore that were exposed to the aqueous environment in the closed and opening conformations (1). Briefly, the sulfhydryl reagent MTSET was allowed to react, in the presence or absence of hypoosmotic shock, with cells expressing MscL channels that contained cysteine substitutions; channel dysfunction was assessed solely by cell viability. Here we evaluate the MTSET-induced functional modifications to these mechanosensitive channel activities by measuring single channel recordings. The observed changes in residue availability in different states, as well as channel kinetics and sensitivity, have allowed us to elucidate the micro-environment encountered for a number of pore residues, thus testing many aspects of previous models and giving a higher resolution of the pore domain and the structural transitions it undergoes from the closed to open state.
Key Words:
MTSET, MscL, SCAM, ion channel gating, mechanosensation, osmoregulation
