2 TYPES OF MECHANOSENSITIVE CHANNELS IN THE ESCHERICHIA-COLI CELL-ENVELOPE - SOLUBILIZATION AND FUNCTIONAL RECONSTITUTION

Mechanosensitive ion channels (MSCs) which could provide for fast osmoregulatory responses in bacteria, remain unidentified as molecular entities. MSCs from Escherichia coli (strain AW740) were examined using the patch-clamp technique, either (a) in giant spheroplasts, (b) after reconstitution by fusing native membrane vesicles with asolectin liposomes, or (c) by reassembly of octylglucoside-solubilized membrane extract into asolectin liposomes. MSC activities were similar in all three preparations, consisting of a large nonselective MSC of 3-nS conductance (in 200 mM KCl) that was activated by high negative pressures, and a small weakly anion-selective MSC of 1 nS activated by lower negative pressures. Both channels appeared more sensitive to suction in liposomes than in spheroplasts. After gel filtration of the solubilized membrane extract and reconstituting the fractions, both large MSC and small MSC activities were retrieved in liposomes. The positions of the peaks of channel activity in the column eluate, assayed by patch sampling of individual fractions reconstituted in liposomes, showed an apparent molecular mass under nondenaturing conditions of about 60-80 kDa for the large and 200-400 kDa for the small MSC. We conclude that (a) the large MSC and the small MSC are distinct molecular entities, (b) the fact that both MSCs were functional in liposomes following chromatography strongly suggests that these channels are gated by tension transduced via lipid bilayer, and (c) chromatographic fractionation of detergent-solubilized membrane proteins with subsequent patch sampling of reconstituted fractions can be used to identity and isolate these MS channel proteins.