The voltage dependence of hEag currents is not determined solely by membrane-spanning domains

Abstract

The ether-à-go-go potassium channels hEag1 and hEag2 are highly homologous. Even though both possess identical voltage-sensing domain S4, the channels act differently in response to voltage. Therefore we asked whether transmembrane domains other than the voltage sensor could contribute to the voltage-dependent behaviour of these potassium channels. For this chimaeras were created, in which each single transmembrane domain of hEag1 was replaced by the corresponding segment of hEag2. The voltage-dependent properties of the chimaeras were analysed after expression in Xenopus laevis oocytes using the two-electrode voltage-clamp method. By this we found, that only the mutations in transmembrane domains S5 and S6 are able to change the voltage sensitivity of hEag1 by shifting the half-activation potential (V 50 ) to values intermediate between the two wild types. Moreover, the presence of Mg 2+ has strong effects on the voltage sensitivity of hEag2 shifting V 50 by more than 50 mV to more positive values. Interestingly, despite the identical binding site Mg 2+ showed only little effects on hEag1 or the chimaeras. Altogether, our data suggest that not only transmembrane spanning regions, but also non-membrane spanning regions are responsible for differences in the behaviour of the hEag1 and hEag2 potassium channels.

The punctate localization of rat Eag1 K+channels is conferred by the proximal post-CNBHD region

“…Despite the presence of about 70% identity in amino acid sequence between the Eag1 and Eag2 K + channel proteins [4,6], the structural bases of their different voltage-gating properties and subcellular localizations have remained largely elusive. Previous biophysical analysis of a series of different chimeras between human Eag1 and Eag2, for example, revealed that the transmembrane regions alone were not sufficient to explain the differences in their gating kinetics and steady-state voltage-dependence [25,26]. In addition, similar to our results here (Figure 8), non-membrane regions per se were found not to determine their gating behaviors [26].…”

Rapid Internalization of the Oncogenic K+ Channel KV10.1

“…The voltage dependence of both constructs was almost identical with a slope of 26.23±4.24 mV for K V 10.1 and 25.63±1.76 mV for K V 10.1-BBS. Also, both constructs displayed rectification at very positive potentials [45].…”

“…We performed two-electrode voltage-clamp recordings to measure current-voltage relationships and channel activation kinetics as described previously [45].…”

Overcoming challenges of HERG potassium channel liability through rational design: Eag1 inhibitors for cancer treatment

“…It is reasonable to assume, however, that the cytoplasmic domains of both channels are functionally similar, since a chimeric channel carrying the PAS domain, C-linker, and CNBHD of HERG on an Eag1 background results in behavior not massively different from wild type Eag1. 28 The mature Eag1 is core glycosylated on Asn-388 and complex glycosylated on Asn-406. Proper glycosylation is required for efficient transport to the plasma membrane, but also enzymatic removal of glycosylation causes a large reduction of the peak current, indicating that the presence of sugar moieties impacts the functional properties of Eag1 channels at the plasma membrane.…”