UNCHARGED S4 RESIDUES AND COOPERATIVITY IN VOLTAGE-DEPENDENT POTASSIUM CHANNEL ACTIVATION

Substitution of the S4 of Shaw into Shaker alters cooperativity in cha nnel activation by slowing a cooperative transition late in the activa tion pathway. To determine the amino acids responsible for the functio nal changes in Shaw S4, we created several mutants by substituting ami no acids from Shaw S4 into Shaker. The S4 amino acid sequences of Shak er and Shaw S4 differ at 11 positions. Simultaneous substitution of ju st three noncharged residues from Shaw S4 into Shaker (V369I, I372L, S 376T; ILT) reproduces the kinetic and voltage-dependent properties of Shaw S4 channel activation. These substitutions cause very small chang es in the structural and chemical properties of the amino acid side ch ains. In contrast, substituting the positively charged basic residues in the S4 of Shaker with neutral or negative residues from the S4 of S haw S4 does not reproduce the shallow voltage dependence or other prop erties of Shaw S4 opening. Macroscopic ionic currents for ILT could be fit by modifying a single set of transitions in a model for Shaker ch annel gating (Zagotta, W.N., T. Hoshi, and R.W. Aldrich. 1994. J. Gen. Physiol 103:321-362). Changing the rate and voltage dependence of a f inal cooperative step in activation successfully reproduces the kineti c, steady state, and voltage-dependent properties of ILT ionic current s. Consistent with the model, ILT Sating currents activate at negative voltages where the channel does not open and, at more positive voltag es, they precede the ionic currents, confirming the existence of volta ge-dependent transitions between closed states in the activation pathw ay. Of the three substitutions in ILT, the I372L substitution is prima rily responsible for the changes in cooperativity and voltage dependen ce. These results suggest that noncharged residues in the S4 play a cr ucial role in Shaker potassium channel gating and that small steric ch anges in these residues can lead to large changes in cooperativity wit hin the channel protein.