CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING |
LOCKING CNGA1 CHANNELS IN THE OPEN AND CLOSED STATE
Anil V. Nair 1, Monica Mazzolini 1, Paolo Codega 1, Alejandro Giorgetti 2 and Vincent Torre 1*
1 International School for Advanced Studies
2 Biocomputing Department of Biochemical Sciences University
* To whom correspondence should be addressed. E-mail: torre{at}sissa.it.
Submitted on August 29, 2005
Revised on September 26, 2005
Accepted on 3 February 2006
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Abstract |
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With the aim of understanding the relation between structure and gating of CNGA1 channels from bovine rod, an extensive cysteine scanning mutagenesis was performed. Each residue from Phe375 to Val424 was mutated into a cysteine one at a time and the modification caused by various sulfhydryl reagents was analyzed. The addition of the mild oxidizing agent copper phenanthroline (CuP) in the open (presence of 1 mM cGMP) or closed state locked the channel in the respective states. A subsequent treatment with the reducing agent DTT restored normal gating fully in the open state and partially in the closed state. This action of CuP was not observed when F380 was mutated into a cysteine in the cysteine free CNGA1 channel and in the double mutant C314S&F380C. These observations suggest that these effects are mediated by the formation of a disulfide bond (S-S) between F380C and the endogenous Cys314 in the S5 segment. It can be rationalized by supposing that during gating the S6 segment rotates anticlockwise - when viewed from the extracellular side - by about 30o.
Key Words:
S6 domain, gating, ionic channels
