PH-Dependent Conformational Changes of KcsA Tetramer and Monomer Probed by Raman Spectroscopy.

Ann-Kathrin Kniggendorf,Oliver Plettenburg,David Schmidt,Bernhard Roth,Carsten Zeilinger

doi:10.3390/ijms20112736

Abstract

KcsA is a tetrameric potassium channel formed out of four identical monomeric subunits used as a standard model for selective potassium transport and pH-dependent gating. Large conformational changes are reported for tetramer and monomer upon gating, and the response of the monomer being controversial with the two major studies partially contradicting each other. KcsA was analyzed as functional tetramers embedded in liposomes and as monomer subunits with confocal Raman microscopy under physiological conditions for the active and the closed channel state, using 532 nm excitation to avoid introducing conformational changes during the measurement. Channel function was confirmed using liposome flux assay. While the classic fingerprint region below 1800 rel. cm−1 in the Raman spectrum of the tetramer was unaffected, the CH-stretching region between 2800 and 3200 rel. cm−1 was found to be strongly affected by the conformation. No pH-dependency was observed in the Raman spectra of the monomer subunits, which closely resembled the Raman spectrum of the tetramer in its active conformation, indicating that the open conformation of the monomer and not the closed conformation as postulated may equal the relaxed state of the molecule.

Highlights

The potassium channel KcsA, originally identified in the bacterium Streptomyces lividans, is a standard model for selective potassium transport and gating function [1,2,3]
We studied KcsA monomers under the same parameters, comparing their spectra with the results obtained for the tetramer
The Raman lines at 2854 and 2962 rel. cm−1 framing the shifted lines are unaffected (Figure 7). In this region, the spectrum of the open channel closely matches the spectrum of the KcsA monomer, which is not affected by the pH conditions at all (Figure 11). This observation agrees with the conformational model for KcsA gating proposed by Hirano et al [22], who described that the four cytoplasmic domains of the KcsA monomers form an oligomer in the closed state, which is destabilized by positively charged amino acids in the open state [22]

Summary

Introduction

The potassium channel KcsA, originally identified in the bacterium Streptomyces lividans, is a standard model for selective potassium transport and gating function [1,2,3]. Each monomer consists of two α-helical transmembrane domains (TM1 and TM2) connected with an extracellular loop leading into a short pore helix and a selective filter structure, responsible for the selectivity of the formed channel to potassium [2,4]. The pore of the channel shows an aqueous cavity with a diameter of 10 Å located below the selectivity filter [2]. Several dynamic analyses by electron spin resonance (ESR) and electrophysiology in combination with site-directed mutagenesis gave a deep understanding of the KcsA channel and the function of its amino acids and domains [11]

Results

Discussion

Conclusion