J. Membrane Biol. 124, 21-32 (1991) 002226319100128W The Journal of MembraneBioloc 9 Spnnger-Verlag New York Inc. 1991 Channel Reconstitution in Liposomes and Planar Bilayers with HPLC-Purified MIP26 of Bovine Lens Lili Shen, Peter Shrager, Stephen J. Girsch, Patricia J. Donaldson, and CamiUo Peracchia Department of Physiology, University of Rochester, Rochester, New York 14642 Summary. The major intrinsic protein (MIP26) of bovine lens membranes, purified by HPLC, was incorporated into liposomes and planar bilayers. Permeability of MIP26 channels was studied in liposomes by a spectrophotometrie osmotic-swelling assay, and channel electrical properties were monitored in planar bilay- ers following liposome fusion. Particle formation in liposomes was determined by freeze fracture. MIP26 channels were perme- able to KC1 and sucrose. In planar bilayers, channel-conduc- tance transitions were observed only after addition of liposomes to both chambers and with voltages greater than -+20 inV. Chan- nel open probability decreased progressively as voltage in- creased, and an open probability of 50% was at 60-80 mV, indi- cating that the channels are voltage dependent. Histograms of single-channel current amplitudes at 80 mV showed a Gaussian distribution that peaked at 10 pA (-120 pS), after subtraction of 1 pA baseline current. Frequency distributions of open and closed times at 80 mV were single exponential functions with time constants of 0.13 and 1.9 sec, respectively. Open time con- stants ranged from 0.1 to 0.3 sec, and closed time constants ranged from I to 7 sec. Cs + did not decrease conductance, but reduced mean open time from 0.2 to 0.038 sec and mean closed time from 1.5 to 0.38 sec. The increase in channel flickering with Cs + occurred in bursts. TEA affected neither conductance nor kinetics. Channel events were also observed in Na + solutions (zero K+). These data indicate that MIP26 channels are not K +- selective channels. Channel characteristics such as: permeability to molecules larger than small ions, conductance greater than 100 pS, long open and closed time constants, etc., are similar to those of gap junction channels. Key Words lens 9 MIP26 9 planar bilayers 9 reconstitution 9 single-channel recording 9 gap junctions 9 ion channels Introduction The eye lens, being an avascular tissue with a nar- row extracellular space and transport enzymes re- stricted almost entirely to the most superficial cells, requires an extensive network of cell-to-cell com- munication for bidirectionally transferring ions and metabolites between deep and superficial fiber cells. Fiber-to-fiber communication is provided by junc- tions with a structure similar to gap junctions that occupy over 50% of the fiber membrane surface (Benedetti et al., 1976). The major intrinsic protein (MIP26) of lens fiber cells is a 28.2-kD component (Gorin et al., 1984) that represents over 60% of the membrane proteins (Benedetti et al., 1976). The amino acid sequence of MIP26 has been inferred from cDNA cloning by Gorin et al. (1984) and found to be extremely hydro- phobic. The folding probability model of the protein depicts six traverses of the lipid bilayer and places both carboxyl and amino termini at the cytoplasmic surface of the membrane (Gorin et al., 1984). In recent years, MIP26 fractions of different de- grees of purity have been successfully incorporated into artificial lipid systems, and the functional prop- erties of the resulting channels have been described (Girsch & Peracchia, 1985; Gooden et al., 1985; Ni- kaido & Rosenberg, 1985; Zampighi, Hall & Kre- man, 1985; Brewer, 1991; Ehring & Hall, 1991; Ehring et al., 1990; Lea & Duncan, 1991). Channels reconstituted into liposomes were found to be per- meable to molecules as large as 1.5 kD (Girsch & Peracchia, 1985; Gooden et al., 1985; Nikaido & Rosenberg, 1985) and were seen to close with Ca 2+ only in the presence of calmodulin (CaM)(Girsch & Peracchia, 1985). Voltage-dependent channels of various conductances were described in planar bi- layers (Zampighi et al., 1985; Ehring et al., 1990; Ehring & Hall, 1991: Lea & Duncan, 1991) and in vesicles formed at the tip of patch pipettes (Brewer, 1990). This paper reports data on permeability and electrical properties of channels reconstituted into liposomes and planar lipid bilayers with high-per- formance liquid chromatography (HPLC)-purified MIP26. The data provide new evidence for the ca- pacity of HPLC-purified MIP26 to form channels permeable to molecules as large as sucrose (tool wt = 342) and show that MIP26 forms only one type of channel in planar bilayers. This is possibly the