High pressure experiments with the porins from the barophile Photobacterium profundum SS9

摘要

Porins are ion channels in the outer membrane of Gram negative bacteria, and their incidence of opening/closing is thought to regulate the composition of the periplasm. In the case of Photobacterium profundum strain SS9, regulation of the expression of two porin genes, ompH and ompL, is determined by hydrostatic pressure. When SS9 is grown at 28MPa the porin OmpH is produced in abundance whilst, conversely, at normal atmospheric pressure OmpL predominates. SS9 grows over the temperature r rage 2-20°C and the pressure range 1-80 MPa, with 28 MPa being optimal. Although OmpH production does not confer tolerance to high pressure (mutants lacking OmpH are not particularly sensitive to pressure), it is reasonable to assume that OmpH functions adequately at high pressure. Equally, there is no obvious reason why OmpL should be pressure-tolerant. Clearly a comparison of these two porins is of considerable interest. Bacterial porins have a distinct structure with which we assume OmpH and OmpL comply. The more familiar porins, such as OmpF and OmpC extracted from E.coli, can be reconstituted in lipid bilayers and hence studied by single channel recording techniques. Reconstitution in liposome bilayers enables patch clamp recordings to be carried out, revealing the porins' opening/closing kinetics, and other properties, in well-defined conditions. Patch clamp recording can be carried out at high pressure and OmpC from E.coli has been studied at pressures up to 90 MPa. It was found that the main effect of pressure was to induce the open state without affecting the porins' conductance. The susceptibility of OmpC to pressure was one factor which stimulated us to undertake the present experiments. More generally high hydrostatic pressures, in the range found in the oceans (up to 100 MPa), exert profound effects on many proteins, both structural and enzymic, and increase the order of lipid bilayers. Experiments with deep-sea animal material have produced some evidence for the adaptation of proteins and bilayers to their normal ambient high pressure and deep-sea bacteria have also revealed their lipid bilayers to be adapted to offset the ordering effect of high pressure. Rather less is known about the effects of high pressure on ion channels in general and thus far no channels (animal or bacterial) which normally function at high pressure have been studied. The availability of OmpH and OmpL has tempted us to undertake high pressure experiments using the same reconstitution procedure which proved successful with Omp C. Here we report limited success and that conversiton of the reconstituton procedure for marine species is required for future progress.