A functional Kv1.2-hERG chimaeric channel expressed in Pichia pastoris

Abstract

Members of the six-transmembrane segment family of ion channels share a common structural design. However, there are sequence differences between the members that confer distinct biophysical properties on individual channels. Currently, we do not have 3D structures for all members of the family to help explain the molecular basis for the differences in their biophysical properties and pharmacology. This is due to low-level expression of many members in native or heterologous systems. One exception is rat Kv1.2 which has been overexpressed in Pichia pastoris and crystallised. Here, we tested chimaeras of rat Kv1.2 with the hERG channel for function in Xenopus oocytes and for overexpression in Pichia. Chimaera containing the S1–S6 transmembrane region of HERG showed functional and pharmacological properties similar to hERG and could be overexpressed and purified from Pichia. Our results demonstrate that rat Kv1.2 could serve as a surrogate to express difficult-to-overexpress members of the six-transmembrane segment channel family.

Determinants of Isoform-Specific Gating Kinetics of hERG1 Channel: Combined Experimental and Simulation Study

“…The recent Cryo-EM structures allowed the structural modeling for open- and closed states of hERG1 channel enabling molecular-level description of the determinants of this apparent isoform-specific differences. Homology models (Wacker et al, 2017 ) and chimera constructs were very useful in the past (Dhillon et al, 2014 ) for understanding structure-function relationships in K+ channels. However, most of the models were focusing on the trans-membrane section of hERG1 channel only (Wacker et al, 2017 ).…”

Getting to the heart of hERG K+ channel gating

“…Two recent studies utilized chimeric approaches with bacterial KcsA (Hausammann & Grütter, ) or rat Kv1.2 (Dhillon et al . ) potassium channels to help stabilize the hERG protein, with the latter reporting a degree of expression and purification of functional chimeric hERG‐Kv1.2 channels in Pichia pastoris . Until we are able to obtain suitable levels of purified protein for X‐ray crystallography or cryo‐electron microscopy experiments, homology modelling and molecular dynamics (MD) simulations remain valuable tools for examining the molecular basis of hERG channel inactivation (Stansfeld et al .…”

A comprehensive study of the voltage gated potassium channel Kv4.3: from functional analysis to molecular dynamics modelling

“…There are different ways to remove these impurities. First, the Nickel ion could be substituted with a Cobalt ion, as in (Dhillon et al, 2015). The latter gives yield to higher specificity than the former at the expense of the protein yield.…”