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
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
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
