Mechanosensitive Channel of Thermoplasma, the Cell Wall-Less Archaea Cloning and Molecular Characterization Anna Kloda and Boris Martinac* Department of Pharmacology, QEII Medical Center, The University of Western Australia, Nedlands, WA 6009 Abstract By using a functional approach of reconstituting detergent-solubilized membrane proteins into liposomes and following their function in patch-clamp experiments, we identified a novel mechanosensitive (MS) channel in the thermophilic cell wall-less archaeon Thermoplasma volca- nium. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the enriched pro- tein fractions revealed a band of approx 15 kDa comparable to MscL, the bacterial MS channel of large conductance. 20 N-terminal residues determined by protein microsequencing, matched the sequence to an unknown open reading frame in the genome of a related species Thermoplasma aci- dophilum. The protein encoded by the T. acidophilum gene was cloned and expressed in Escherichia coli and reconstituted into liposomes. When examined for function, the reconstituted protein exhibited properties typical of an MS ion channel: 1) activation by negative pressure applied to the patch-clamp pipet, 2) blockage by gadolinium, and 3) activation by the anionic amphipath trini- trophenol. In analogy to the nomenclature used for bacterial MS channels, the MS channel of T. aci- dophilum was termed MscTA. Secondary structural analysis indicated that similar to MscL, the T. acidophilum MS protein may have two transmembrane domains, suggesting that MS channels of thermophilic Archaea belong to a family of structurally related MscL-like ion channels with two membrane-spanning regions. When the mscTA gene was expressed in the mscL – knockout strain and the MscTA protein reconstituted into liposomes, the gating of MscTA was characterized by very brief openings of variable conductance. In contrast, when the mscTA gene was expressed in the wild-type mscL + strain of E. coli, the gating properties of the channel resembled MscL. However, the channel had reduced conductance and differed from MscL in its kinetics and in the free energy of activation, suggesting that MscTA and MscL can form functional complexes and/or modulate each other activity. Similar to MscL, MscTAexhibited an increase in activity in liposomes made of phospholipids having shorter acyl chain, suggesting a role of hydrophobic mismatch in the function of prokaryotic MS channels. Index Entries: MS channels; patch clamp; gene cloning; Archaea; MscL; hydrophobic mismatch; amphipaths; liposome reconstitution. * Author to whom all correspondence and reprint requests should be addressed. Department of Pharmacology, QEII Medical Center, The University of Western Australia, Crawley, WA6009, Australia. E-mail: bmartinac@receptor.pharm. uwa.edu.au Cell Biochemistry and Biophysics 321 Volume 34, 2001 ORIGINAL ARTICLE © Copyright 2001 by Humana Press Inc. All rights of any nature whatsoever reserved. 1085-9195/01/34/321–347/$16.75