Control of Outer Vestibule Dynamics and Current Magnitude in the Kv2.1 Potassium Channel

Abstract

In Kv2.1 potassium channels, changes in external [K+] modulate current magnitude as a result of a K+-dependent interconversion between two outer vestibule conformations. Previous evidence indicated that outer vestibule conformation (and thus current magnitude) is regulated by the occupancy of a selectivity filter binding site by K+. In this paper, we used the change in current magnitude as an assay to study how the interconversion between outer vestibule conformations is controlled. With 100 mM internal K+, rapid elevation of external [K+] from 0 to 10 mM while channels were activated produced no change in current magnitude (outer vestibule conformation did not change). When channels were subsequently closed and reopened in the presence of elevated [K+], current magnitude was increased (outer vestibule conformation had changed). When channels were activated in the presence of low internal [K+], or when K+ flow into conducting channels was transiently interrupted by an internal channel blocker, increasing external [K+] during activation did increase current magnitude (channel conformation did change). These data indicate that, when channels are in the activated state under physiological conditions, the outer vestibule conformation remains fixed despite changes in external [K+]. In contrast, when channel occupancy is lowered, (by channel closing, an internal blocker or low internal [K+]), the outer vestibule can interconvert between the two conformations. We discuss evidence that the ability of the outer vestibule conformation to change is regulated by the occupancy of a nonselectivity filter site by K+. Independent of the outer vestibule-based potentiation mechanism, Kv2.1 was remarkably insensitive to K+-dependent processes that influence current magnitude (current magnitude changed by <7% at membrane potentials between −20 and 30 mV). Replacement of two outer vestibule lysines in Kv2.1 by smaller neutral amino acids made current magnitude dramatically more sensitive to the reduction in K+ driving force (current magnitude changed by as much as 40%). When combined, these outer vestibule properties (fixed conformation during activation and the presence of lysines) all but prevent variation in Kv2.1 current magnitude when [K+] changes during activation. Moreover, the insensitivity of Kv2.1 current magnitude to changes in K+ driving force promotes a more uniform modulation of current over a wide range of membrane potentials by the K+-dependent regulation of outer vestibule conformation.

Allosteric effects of external K+ ions mediated by the aspartate of the GYGD signature sequence in the Kv2.1 K+ channel

“…The most favorable conformation now involves an H-bond between the side chains of E80 and C81 occupancy of the sites in the filter and inducing conformational changes in the external vestibule through an inter-subunit H-bond network. These data add further support for the idea that the external vestibule and selectivity filter are dynamic structures [18,33,38,57,75] critically involved in many aspects of channel function. Fig.…”

“…D378 in Kv2.1 may be involved in some of these phenomena. External K + ions can induce conformational rearrangements of the external vestibule in Kv2.1 that affect TEA binding [33] and K + current magnitude [38], and these effects have been suggested to be mediated by a low-affinity, external K + -binding site associated with the selectivity filter [68]. C-type inactivation [25,28,69] has been shown to be antagonized by external K + ions [70].…”

“…In Kv2.1, external K + ions binding to a site in the selectivity filter determine the conformation of the external vestibule [33] and thereby affect the affinity and efficacy of tetraethylammonium (TEA) [34,35] inactivation rate [36,37] and the macroscopic K + conductance [38]. A strict coupling between gating and permeation has also been proposed to explain subconductance behavior in single-channel recordings [39,40].…”

Characterization of chromanol 293B-induced block of the delayed-rectifier K+ current in heart-derived H9c2 cells

“…Therefore, its inhibition of I K might result from open channel block. It also remains to be determined to what extent this compound may have a higher affinity for the open conformation of K + channels than for the closed conformation without evoking open channel block, because the outer vestibule of the Kv2.1 channel might interconvert between the two conformations (Andalib et al, 2002).…”

“…The expression of Kv2.1 mRNA in heart could be altered under certain pathological conditions associated with arrhythmia (Xu et al, 1999;Zhou et al, 2003;Huang et al, 2000). Moreover, the Kv2.1-encoded K + channels could be important under conditions of high frequency firing of neurons (Klemic et al, 1998;Du et al, 2000;Andalib et al, 2002), and have been found to be linked to a mechanism through which the apoptotic signaling cascade occurs in cortical neurons (Pal et al, 2003) and in H9c2 cells (Ekhterae et al, 2003). This cell line is thus thought to be a useful model with which to study the biophysical and pharmacological properties of Kv2.1 channel.…”