SIDE-CHAIN ACCESSIBILITIES IN THE PORE OF A K-SPECIFIC REAGENTS AFTERCYSTEINE-SCANNING MUTAGENESIS( CHANNEL PROBED BY SULFHYDRYL)

To gain insight into the secondary structure of the ion conduction pat hway of a voltage-gated K+ channel, we used sulfhydryl-specific reagen ts of different diameters to probe amino acid side-chain accessibiliti es in the pore of the channel after cysteine-substitution mutagenesis. We identified five positions at which modified amino acid side chains are accessible from the aqueous lumen of the external channel vestibu le. Covalent coupling of the 2-trimethylammonium-thioethyl group to cy steine thiols leads to position-dependent current reduction, suggestin g a gradual narrowing of the pore. The fact that the modified side cha ins of two adjacent amino acids are reactive is not compatible with th e ion conduction pathway forming a regular beta-pleated sheet at these positions. The smaller thiol reagent Cd2+ reacts with modified side c hains that are also accessible to the larger (2-trimethylammoniumethy l)methanethiosulfonate (MTSET). Our results imply that the outer vesti bule of a potassium-selective ion channel narrows over a short distanc e of three amino acids near a position where a regular beta-structure is unlikely.