Zeilinger, Carsten: MJK2, a K+Channel from M. Jannaschii Mediates pH Dependent Potassium Transport Activity. In: Journal of Physical Chemistry & Biophysics 4 (2014), Nr. 5. DOI: dx.doi.org/10.4172/2161-0398.1000156
Zusammenfassung: | |
MjK2 was expressed in E. coli cells as a fusion protein containing N-or C-terminal an antibody binding site and a histidinehexamer. The C-terminal tagged fusion protein allows the expression and purification of an extra soluble RCK domain at p34 kDa, whereas this additional RCK domain was lost when the N-terminal tagged construct was used. Upon removal of the fusion peptide from the purified N-terminal tagged channel monomer, MjK2 occurred as a stable tetramer when incubated with synthetic lipid. The channel activity was studied after reconstitution into liposomes by single channel recording or by an optical assay with the potassium sensing dye, PBFI. First the channel function was improved by single channel recording. Single channel recording confirmed the pH dependence of the channel activity with single channel conductances of 42, 70, 85 and 202 pS and indicated that a functional K+ channel was formed. To study the function of the reconstituted MjK2 activity in an optical assay the potassium release was initiated when the external BaCl2 block was compensated by addition of EDTA. The release of potassium was mediated by reconstituted MjK2 at low pH or by the presence of internal calcium at high pH. MgCl2 had no or weak effect, whereas cAMP at low pH caused a complete loss of potassium during the preparation. Alignments studies revealed that MjK2 has different structural features in the channel pore and the RCK composition and therefore a different function can be expected. Amino acid sequence and structural alignments showed that a Ca2+ binding site and a typical nucleotide-binding site is not present in the RCK domain of MjK2 and therefore a different behavior could be expected. In addition a lysine reach linker region as found in human sperm K+ channels hslo1 and hslo3can play similar role in the gating behavior. | |
Lizenzbestimmungen: | CC BY 4.0 Unported |
Publikationstyp: | Article |
Publikationsstatus: | publishedVersion |
Erstveröffentlichung: | 2014 |
Die Publikation erscheint in Sammlung(en): | Naturwissenschaftliche Fakultät |
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