Effect of High Hydrostatic Pressure on the porin OmpC from E.coli

 

A.G. Macdonald and B. Martinac

 

University of Aberdeen, Department of Biomedical Sciences, Zoology Building, Aberdeen, AB24 2TZ, Scotland, UK, a.macdonald@abdn.ac.uk and

Department of Pharmacology, University of Western Australia, Nedlands, WA6907, Australia, bmartinac@receptor.pharm.uwa,edu.au

 

Introduction Porins are channels in the outer membrane of Gram-negative bacteria and their physiological role is to provide a permeability path for small molecules, generally less than 600 Daltons. OmpC is a well characterized porin [1] and can be extracted and reconstituted in either planar bilayers or liposome bilayers, which enable its permeability and gating kinetics to be studied. The liposome technique has been used here, because it allows patch clamp recordings to be carried out at high hydrostatic pressure, using the procedure described in [2].

Porins are trimeric membrane proteins whose structure is well known, and whose response to high pressure should be capable of detailed interpretation. Pressure may well reveal something of the molecular changes which underlie the gating processes. Furthermore pressure may harm or benefit bacteria by affecting the physiological function of porins, by modifying their permeability/selectivity and gating.

 

Materials & Methods OmpC was extracted from E. coli cultures, following the method described in [3]. Reconstitution into liposomes and their subsequent osmotic swelling followed the procedure in [4]. Standard patch clamp methods were used to obtain inside-out patches which were subjected to pressure of up to 95 MPa at 22 3C, following [2]. The various solutions used were: A. in mM KCl, 200; MgCl2 40; BIS-TRIS (Sigma) 5, pH6.1 B. KCl, 150; K-EDTA 0.1; CaCl2, 0.01, BIS-TRIS, 5, pH7.2 C. as B, buffered with Trizma Hcl/ Base (Sigma) 5, pH 9.1.

 

Results Control Conditions The number of porins in the patch was variable, but was probably more than 10. Applying potentials of + or - 100 mV, or of a lesser voltage, for 20-30 secs, invariably elicited "micro-activity", defined here as brief pulses of current (1 pA) occurring at a frequency of about 1s-1, superimposed on a "baseline" [1] current. The magnitude of this current depended on the number of permanently open porins in the patch. The current fluctations corresponded to an individual channel (monomer) opening or closing. Such background activity was seen in symmetrical solutions A or B; a few experiments in C revealed a greater level of background activity.

High Pressure The baseline current was often increased on compression, reversibly, but there was no immediate effect on the micro activity. In solution A there occurred one or more bursts of current corresponding to multiple channel openings, (up to 100 pA) many minutes after the application of 30 MPa. A different pressure profile was mostly used with solution B, comprising an initial 30 MPa step followed 10 minutes later by a step up to 82 MPa. Invariably, the higher pressure caused bursts of channel openings. A similar pattern was seen with solution C.

 

References:

[1] Delcour, A.H. (1997) FEMS Microbiology letters 151, 115-123.

[2] Macdonald, A.G. (1997) Biophys. J. 73, 1866-1873.

[3] Le Dain, A.C., Häse, C.C., Tommassen, J. and Martinac, B. (1996) EMBO Journal 15, 3524-3528.

[4] Delcour, A.H., Martinac, B., Adler, J. and Kung, C. (1989) Biophys. J. 56, 631-636.

 

 

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