Biochimica et Biophysica Acta, 1024 (1990) 111-121 111 Elsevier BBAMEM 74821 Ion channel activities in the Escherichia coli outer membrane Matthew Buechner 1, Anne H. Delcour a, Boris Martinac 2, Julius Adler 1 and Ching Kung 2 t Departments of Biochemistry and Genetics, and 2 Laboratory of Molecular Biology and Department of Genetics, Unioersity of Wisconsin, Madison, WI (U.S.A.) (Received 10 October 1989) Key words: Porin; Ion channel; Outer membrane; Patch-clamp; (E. coil) The electrical properties of Escherichia coli cells were examined by the patch-clamp technique. Giant cells or giant spheroplasts were generated by five different methods. By electron micrographic and other criteria we determined that the patches are most likely from the outer membrane. We regularly observed currents through at least two types of channels in this membrane. The first current is mechanosensitive and voltage-dependent, and can be observed in single gene mutants of the known major porins (ompF, ompC, phoE, lamB); this channel may represent a minor porin or a new class of outer membrane protein. The possible identity of the second, voltage-sensitive channel with one of the known outer membrane proteins is being explored. The high-resistance seals consistently formed on these patches and the presence of gated ion channels suggest that most of the pores of the outer membrane are not statically open, as commonly held, but are closed at rest and may be openable by physiological stimuli. Introduction Gram-negative bacteria are enclosed by an outer membrane and an inner (cytoplasmic) membrane, sep- arated by a peptidoglycan cell wall and a periplasmic space [1]. The outer membrane is considered to be a barrier for macromolecules, but not for small hydro- philic solutes. In Escherichia coil, solutes smaller than 600 Da diffuse through the two major porins, OmpF and OmpC, proteins which form nonspecific channels across the outer membrane [2-6]. Different porins and other proteins also exist (LamB, PhoE, NmpC, Tsx, Fhu, Fep) for transport of specific solutes [6-9]. Each bacterium is estimated to have 105 porins in its outer membrane [10]. The inner membrane acts as a selectively permeable barrier to allow passage only of compounds such as amino acids, sugars, and ions, for which there are transport mechanisms [11]. Several membrane proteins exist for conversion of the protonmotive force into sodium and potassium gradients, as well as for trans- port of organic molecules, synthesis of ATP, and for driving the flagellar rotary motor. It is expected that any small-conductance bacterial ion channels, such as Correspondence: J. Adler, Department of Biochemistry and Genetics, University of Wisconsin, Madison, WI 53706, U.S.A. might be used for sensory transduction, would also be located in this membrane. We have explored the use of the patch-clamp tech- nique [12] to study the electrophysiological properties of ion channels of native bacterial membranes [13]. This method is applicable to E. coli, although we must use giant cells or giant spheroplasts (cells in which the peptidoglycan is partially dissolved). We found activi- ties of ion channels that are clearly gated by mechanical forces and/or by voltage, in manners similar to those of eucaryotic ion channels. Here we have tried to de- termine whether our patches are formed on the outer or inner membrane by variations in the method of spheroplast and cell preparation, electron microscopic observation, partial purification of channel activities, and treatment of the patches with lysozyme. Materials and Methods Chemicals and growth media Growth media and chemicals were obtained from standard sources as reported previously [13]. Luria- Bertani medium contains 1% Bacto-Tryptone, 0.5% yeast extract, and 1% NaC1. Modified Luria-Bertani medium is the same except that only 0.5% NaC1 is used. Patch- clamp solutions were buffered by dilution of a 1 M solution of 4-(2-hydroxyethyl)-l-piperazineethanesul- fonic acid (Hepes) brought to pH 7.2 with concentrated 0005-2736/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)