COMMUNICATION Voltage-gating of Escherichia coli Porin: A Cystine-scanning Mutagenesis Study of Loop 3 Graeme Bainbridge 1 , Hamid Mobasheri 2 , Geoffrey A. Armstrong 1 Edward J. A. Lea 2 and Jeremy H. Lakey 1 * 1 Department of Biochemistry and Genetics, The Medical School, University of Newcastle Upon Tyne, NE2 4HH, UK 2 School of Biological Sciences University of East Anglia, Norwich, Norfolk, NR4 7TJ UK Porins, such as Escherichia coli OmpF, provide the only reported example of a voltage-gated channel where the three-dimensional structure is known to high resolution. Mutations that affect voltage-gating are clus- tered around the eyelet region, which is a mid-channel constriction caused by a polypeptide loop (L3) folding inside the lumen of this b-bar- rel pore. These data, combined with molecular dynamics simulations, indicate that voltage-gating may involve L3 displacement. We have con- structed six double cysteine OmpF mutants, ®ve of which form disul- phide bonds ®xing L3 in the conformation determined by X-ray crystallography. These channels have altered single-channel conductances but unimpaired voltage-gating. The data show that L3 movement is not required for voltage-gating. # 1998 Academic Press Limited Keywords: porin; OmpF; planar bilayers; disulphide; voltage-gating *Corresponding author Porins of the outer membrane of Gram-negative bacteria are currently the only transmembrane channels with known high-resolution 3D structures (Cowan et al., 1992; Hirsch et al., 1997; Kreusch et al., 1994; Przybylski et al., 1996; Schirmer et al., 1995; Weiss & Schultz, 1992). The non-selective porins are water-®lled pores which permit free dif- fusion of molecules smaller than 600 Da into and out of the periplasm, whereas selective porins, e.g. maltoporin or Tsx, facilitate the free diffusion of de®ned classes of molecules such as maltodextrins and nucleosides, respectively. They all form tri- mers, and the non-selective group consists of 16-stranded b-barrels with each monomer forming a discrete pore. Within each pore a long polypep- tide loop (L3) runs along one side of the barrel wall and narrows the pore to create the ``eyelet'' region. These porins have a strong transverse elec- tric ®eld across the eyelet region generated by basic residues on the barrel wall and acidic resi- dues and peptide carbonyl groups on L3 (Karshikoff et al., 1994). Porins form large ion-conducting channels in planar lipid bilayer reconstitutions and they mostly display symmetrical voltage-gating, that is, they close in response to applied voltages of either polarity which exceed a threshold value. Although somewhat variable (Buehler et al., 1991; Lakey & Pattus 1989) this threshold voltage may be de®ned by either an equilibrium value, for which we use the term V i (Dargent et al., 1986; Schindler & Rosenbusch, 1978), or as the result of a continuous ascending voltage ramp (Lakey & Pattus, 1989), here referred to as V c (Saint et al., 1996b). Since the voltage ramp does not allow equilibrium to be reached, V c > V i (Saint et al., 1996b). Another point of reference is the voltage required to close all the channels present in the membrane, V f (V f > V c > V i ). The three major porins of Escherichia coli, PhoE, OmpF and OmpC, display V c values of 135 mV (Van Gelder et al., 1997), 145 to 155 mV (Saint et al., 1996a; Van Gelder et al., 1997) and >200 mV (Buehler et al., 1991; Lakey et al., 1991), respectively. The structure of OmpC is not known but it shares 64% and 62% residue identity with OmpF and PhoE, respectively, with the level of identity rising when only the b-barrel core and L3 region are considered. Hence their backbone atoms should superimpose with an RMS of <2.0 A Ê (Chothia & Lesk, 1986). Porins also form a sequence superfamily so that data from most mem- bers can be usefully compared (Jeanteur et al., 1991). Voltage-gating has not been observed in vivo (Sen et al., 1988) and its role in bacterial physiology is not clear. In patch-clamped outer membranes, porins also show many rapid channel events which are voltage sensitive, especially in the pre- sence of molecules such as cadaverine (Dela Vega & Delcour, 1995). Porins from Neisseria can insert J. Mol. Biol. (1998) 275, 171±176 0022±2836/98/020171±06 $25.00/0/mb971474 # 1998 Academic Press Limited