Transport mechanism of Na+/H+ exchange: Identification of amino acid residues essential for the function of the NhaB Na+/H+ antiporter from Klebsiella pneumoniae
Duration: 24 months
Finance source: State budget, contracting authority: Romanian National Authority for Scientific Research and
Innovation, CNCS – UEFISCDI
Budget: 250000 RON
Project start: 02.05.2018
Project end: 30.04.2020
Living cells require a strict control of their intracellular conditions in order to ensure continued survival. Integral membrane proteins that
exist in all living organisms, the Na+/H+ antiporters, are responsible for the regulation of the internal Na+ concentration, intracellular pH and cellular volume. The best understood Na+/H+ antiporters belong to the cation proton antiporter (CPA) superfamily, which includes, among others, the human NHE- and NHA-type antiporters as well as bacterial NhaA- and NhaP-type antiporters. Crystal structures of NhaA- and NhaP-type antiporters have been determined, amino acid residues essential for their transport function have been identified, and their activity has been characterized in detail.
Much less is known about non-CPA antiporters, such as the NhaB-type antiporters, although they have been shown to be involved in bacterial survival and virulence. In a separate project, we are undergoing the first complete electrophysiological characterization of a NhaB Na+/H+ antiporter from the pathogenic bacterium Klebsiella pneumoniae (KpNhaB). However, a better understanding of NhaB proteins requires more in-depth studies of these antiporters.
To that end, the current project aims to perform mutagenesis of several amino acid residues present in the transmembrane helices of KpNhaB and conserved in the NhaB family. We will observe the consequences of these mutations on the NaNa+/H+ exchange function of KpNhaB by characterizing mutant KpNhaB variants using solid supported membrane (SSM)-based electrophysiology, an experimental technique particularly adapted for the study of prokaryotic transport proteins. The study will reveal valuable information about the transport mechanism of NhaB antiporters and will serve as the basis for future studies on NhaB antiporters. Furthermore, it will contribute to the establishment of KpNhaB as a model system for the characterization of the NhaB family.
Lecturer Dr. Octavian Călinescu – Project Director
Prof. Dr. Constanța Ganea – Mentor
Results (phase 1)
- Obtaining plasmids containing mutants of KpNhaB (ACHIEVED)
- Expression of the mutants in E. coli (ACHIEVED)
Results (phase 2)
- Investigate function of KpNhaB N32A (ACHIEVED)
- Investigate function of KpNhaB D146A (ACHIEVED)
- Investigate function of KpNhaB D146E (ACHIEVED)
- Investigate function of KpNhaB D404A (ACHIEVED)
- Investigate function of KpNhaB D404E (ACHIEVED)
- Investigate function of KpNhaB N396A (ACHIEVED)
Results (phase 3)
- Kinetic analysis of the mutants (ACHIEVED)
- Publishing the project’s results (ACHIEVED)
The reports shown here are redacted versions that do not contain all the data generated in the project. For the full report you can contact the project director at octavian.calinescu (at) umfcd.ro
1. Patino-Ruiz, M., Ganea, C., Fendler, K., Călinescu, O., “The Na+/H+ Exchanger NhaB from Klebsiella pneumoniae – A Model System for Electrophysiological Studies.”, 15th National Conference of Biophysics, 07-10 September 2018, Bucharest, Romania.
2. Patino-Ruiz, M., Ganea, C., Fendler, K., Călinescu, O., “Mutational study of the NhaB Na+/H+ exchanger from Klebsiella pneumoniae”, FEBS Advanced Lecture Course Biochemistry of Membrane Proteins – Structure, Trafficking, Regulation, 25-30 August 2019, Budapest, Hungary.
1. Patino-Ruiz, M., Dwivedi, M., Călinescu, O., Karabel, M., Padan, E., Fendler, K. 2019. Replacement of Lys-300 with a glutamine in the NhaA Na+/H+ antiporter of Escherichia coli yields a functional electrogenic transporter, J. Biol. Chem. 294, 246-256.
2. Patino-Ruiz, M., Fendler, K., Călinescu, O. 2019. Mutation of two key aspartate residues alters stoichiometry of the NhaB Na+/H+ exchanger from Klebsiella pneumoniae, Sci. Rep. 9, 15390.