Uncharged S4 Residues and Cooperativity in Voltage-dependent Potassium Channel Activation

Abstract

Substitution of the S4 of Shaw into Shaker alters cooperativity in channel activation by slowing a cooperative transition late in the activation pathway. To determine the amino acids responsible for the functional changes in Shaw S4, we created several mutants by substituting amino acids from Shaw S4 into Shaker. The S4 amino acid sequences of Shaker and Shaw S4 differ at 11 positions. Simultaneous substitution of just three noncharged residues from Shaw S4 into Shaker (V369I, I372L, S376T; ILT) reproduces the kinetic and voltage-dependent properties of Shaw S4 channel activation. These substitutions cause very small changes in the structural and chemical properties of the amino acid side chains. In contrast, substituting the positively charged basic residues in the S4 of Shaker with neutral or negative residues from the S4 of Shaw S4 does not reproduce the shallow voltage dependence or other properties of Shaw S4 opening. Macroscopic ionic currents for ILT could be fit by modifying a single set of transitions in a model for Shaker channel 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 final cooperative step in activation successfully reproduces the kinetic, steady state, and voltage-dependent properties of ILT ionic currents. Consistent with the model, ILT gating currents activate at negative voltages where the channel does not open and, at more positive voltages, they precede the ionic currents, confirming the existence of voltage-dependent transitions between closed states in the activation pathway. Of the three substitutions in ILT, the I372L substitution is primarily responsible for the changes in cooperativity and voltage dependence. These results suggest that noncharged residues in the S4 play a crucial role in Shaker potassium channel gating and that small steric changes in these residues can lead to large changes in cooperativity within the channel protein.

Atypical Gating Of M-Type Potassium Channels Conferred by Mutations in Uncharged Residues in the S4Region of KCNQ2 Causing Benign Familial Neonatal Convulsions

“…S 4 hydrophobic residues are involved in K ϩ channel voltagedependent gating (Lopez et al, 1991;McCormack et al, 1991), and it has been proposed that the gating differences between Shaker (K v 1) and Shaw (K v 3) channels are mainly accounted for by sequence differences with respect to S 4 uncharged amino acids (Smith-Maxwell et al, 1998). Uncharged amino acids also occupy the positions corresponding to A196 and L197 in the K v 1.2 voltage-gated K ϩ channel (Fig.…”

Cloning and Functional Characterization of the Smooth Muscle Ether-a-go-go-related Gene K+ Channel

“…The S4 segment is proposed to function as a voltage sensor with positively charged residues critical in voltage-dependent transitions for activation (36). Recent studies by Smith-Maxwell et al (37,38) suggest that single conserved uncharged amino acid substitution within the S4 region may be important in cooperativity and voltage dependence of Shaker K ϩ channels. These authors demonstrated that substitution of leucine for isoleucine (that differ in the attachment of a methyl group) resulted in changes in the gating charge movement.…”

Extracellular Linkers Completely Transplant the Voltage Dependence from Kv1.2 Ion Channels to Kv2.1

“…1 implies that the correlation between j out and V 1/2 depends on the variability of j in þ G 0 /F. This component, the residual field potential, has been shown to influence V 1/2 in various channels: for instance, electroneutral mutations at the interior of the Shaker Kv channel (not expected to affect j out ) have considerable effects on V 1/2 (49)(50)(51)(52). Because the extracellular linkers L3/4, L5/P, and LP/6 were sufficient to transfer the alteration in j out we analyzed the contribution of the different components in more detail, assuming that the unchanged parts would remain relatively constant (Fig.…”