Characterization of the C-terminal Domain of a Potassium Channel from Streptomyces lividans (KcsA)

Zhiguang Yuchi,Yongfang Zhu,Victor P.T. Pau,Quyen Q. Hoang,Daniel S.C. Yang

doi:10.1074/jbc.m703277200

Abstract

KcsA, a potassium channel from Streptomyces lividans, is a good model for probing the general working mechanism of potassium channels. To date, the physiological activator of KcsA is still unknown, but in vitro studies showed that it could be opened by lowering the pH of the cytoplasmic compartment to 4. The C-terminal domain (CTD, residues 112-160) was proposed to be the modulator for this pH-responsive event. Here, we support this proposal by examining the pH profiles of: (a) thermal stability of KcsA with and without its CTD and (b) aggregation properties of a recombinant fragment of CTD. We found that the presence of the CTD weakened and enhanced the stability of KcsA at acidic and basic pH values, respectively. In addition, the CTD fragment oligomerized at basic pH values with a transition profile close to that of channel opening. Our results are consistent with the CTD being a pH modulator. We propose herein a mechanism on how this domain may contribute to the pH-dependent opening of KcsA.

Highlights

Permeation of ions across cellular membranes is essential to life, but it is energetically unfavorable due to the dielectric barrier formed by the lipidic components of the membrane
Crystal structure of a truncated KcsA (PDB id 1K4C) showed two transmembrane ␣-helices separated by a P loop in each subunit [11, 12]
The N-terminal and C-terminal domains (CTD)2 are absent in the crystal structure (Protein Data Bank id 1K4C), they are predicted to face toward the cytosol [14, 16]

Summary

EXPERIMENTAL PROCEDURES

Material—Electrophoresis setups were purchased from BioRad Laboratories Ltd. (Ontario, Canada). Material—Electrophoresis setups were purchased from BioRad Laboratories Ltd. Taq polymerase, Pfu polymerase, and T4 ligase were purchased from Fermentas Canada Inc. HiTrap chelating HP and HiTrap heparin columns and pET28a. Characterization of the C-terminal Domain of KcsA with MWCO: 50,000 were bought from Avanti Polar Lipid, Inc. (Alabaster, Al) and VWR (Ontario, Canada), respectively. Cloning of KcsA (rKcsA), C-terminal His-tagged KcsA (chKcsA), and N-terminal His-tagged rCTD of KcsA—The DNA coding for

KcsA was amplified by PCR with

The DNA coding for recombinant

RESULTS

DISCUSSION