THE SPONTANEOUS GATING ACTIVITY OF OMPC PORIN IS AFFECTED BY MUTATIONS OF A PUTATIVE HYDROGEN-BOND NETWORK OR OF A SALT BRIDGE BETWEEN THE L3 LOOP AND THE BARREL

Porins are trimeric channel-forming proteins of the outer membrane of Escherichia coli, Each subunit contains 16 P-strands forming a transme mbrane beta-barrel whose pore is constricted by the third extracellula r loop (L3). We investigated the effects of site-directed mutations at two critical regions of the OmpC porin: (i) the D315A mutation target s a key component of a putative hydrogen bond network linking the L3 l oop to the adjacent barrel wall and (ii) the D118Q, R174Q and R92Q mut ations target putative salt bridges at the root of the L3 loop. We pur ified the outer membrane fractions obtained from each mutant and recon stituted them in liposomes suitable for electrophysiology, Patch clamp experiments showed that the frequency of spontaneous transitions betw een open and closed states is increased in the D315A, D118Q and R92Q m utants but unchanged in the R174Q mutant. These transitions are not dr iven by transmembrane voltage changes and represent the thermal oscill ations between functionally distinct conformations. The asymmetric vol tage-dependent inactivation of the channels is not affected by the mut ations, however, suggesting different molecular mechanisms for the spo ntaneous and voltage-dependent gating processes. We propose that the p ositioning or flexibility of the L3 loop across the pore, as governed by the putative hydrogen-bond network and a salt bridge, play a role i n determining the frequency of spontaneous channel gating.