P-LOOP FLEXIBILITY IN NA-CYSTEINE AND DOUBLE-CYSTEINE REPLACEMENTS( CHANNEL PORES REVEALED BY SINGLE)

Replacement of individual P-loop residues with cysteines in rat skelet al muscle Na+ channels (SkM1) caused an increased sensitivity to curre nt blockade by Cd2+ thus allowing detection of residues lining the por e. Simultaneous replacement of two residues in distinct P-loops create d channels with enhanced and reduced sensitivity to Cd2+ block relativ e to the individual single mutants, suggesting coordinated Cd2+ bindin g and cross-linking by the inserted sulfhydryl pairs. Double-mutant ch annels with reduced sensitivity to Cd2+ block showed enhanced sensitiv ity after the application of sulfhydryl reducing agents. These results allow identification of residue pairs capable of approaching one anot her to within less than 3.5 Angstrom. We often observed that multiple consecutive adjacent residues in one P-loop could coordinately bind Cd 2+ with a single residue in another P-loop. These results suggest that , on the time-scale of Cd2+ binding to mutant Na+ channels, P-loops sh ow a high degree of flexibility.