Constitutive Activation of the Shaker Kv Channel

Abstract

In different types of K+ channels the primary activation gate is thought to reside near the intracellular entrance to the ion conduction pore. In the Shaker Kv channel the gate is closed at negative membrane voltages, but can be opened with membrane depolarization. In a previous study of the S6 activation gate in Shaker (Hackos, D.H., T.H. Chang, and K.J. Swartz. 2002. J. Gen. Physiol. 119:521–532.), we found that mutation of Pro 475 to Asp results in a channel that displays a large macroscopic conductance at negative membrane voltages, with only small increases in conductance with membrane depolarization. In the present study we explore the mechanism underlying this constitutively conducting phenotype using both macroscopic and single-channel recordings, and probes that interact with the voltage sensors or the intracellular entrance to the ion conduction pore. Our results suggest that constitutive conduction results from a dramatic perturbation of the closed-open equilibrium, enabling opening of the activation gate without voltage-sensor activation. This mechanism is discussed in the context of allosteric models for activation of Kv channels and what is known about the structure of this critical region in K+ channels.

Tetraphenylporphyrin derivative specifically blocks members of the voltage-gated potassium channel subfamily Kv1

“…The choice of this mutation originated from a report that the analogous mutation renders the Shaker channel constitutively open. 23 Indeed, Kv1.3 P427D channels showed a comparable voltage-independent gating behavior (Fig. 5A).…”

Hydrophobic Interactions as Key Determinants to the KCa3.1 Channel Closed Configuration

“…2ϩ -Constitutive activation of the Shaker channel following the mutation of Pro 475 (Ala 283 in KCa3.1) to Asp has been reported previously (41). It was suggested that Pro 475 was located in a restricted hydrophobic environment in the closed state, so that substituting the Pro by a more hydrophilic residue strongly affected residue packing at the bundle crossing region resulting in an unstable closed configuration (41).…”

“…It was suggested that Pro 475 was located in a restricted hydrophobic environment in the closed state, so that substituting the Pro by a more hydrophilic residue strongly affected residue packing at the bundle crossing region resulting in an unstable closed configuration (41). Similarly, a yeast screening analysis of the GIRK2 channel has revealed that the substitution FIGURE 9.…”

Principles of conduction and hydrophobic gating in K + channels

“…Third, alteration of cavity hydrophobicity shifts the open/closed equilibrium (15,(37)(38)(39)(40)(41)(42). Substitution of Shaker Pro407 by hydrophilic residues (for example, Pro407Asp) yields constitutively open channels, whereas hydrophobic mutants, such as Val410Trp, are constitutively closed.…”