Channel reconstitution in liposomes and planar bilayers with HPLC-purified MIP26 of bovine lens.
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 spectrophotometric osmotic-swelling assay, and channel electrical properties were monitored in planar bilayers following liposome fusion. Particle formation in liposomes was determined by freeze fracture. MIP26 channels were permeable to KCl and sucrose. In planar bilayers, channel-conductance transitions were observed only after addition of liposomes to both chambers and with voltages greater than +/- 20 mV. Channel open probability decreased progressively as voltage increased, and an open probability of 50% was at 60-80 mV, indicating that the channels are voltage dependent. Histograms of single-channel current amplitudes at 80 mV showed a Gaussian distribution that peaked at 10 pA (approximately 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 constants ranged from 0.1 to 0.3 sec, and closed time constants ranged from 1 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.
Shen
L
L
Department of Physiology, University of Rochester, New York 14642.
Shrager
P
P
Girsch
S J
SJ
Donaldson
P J
PJ
Peracchia
C
C
D000818:Animals; D020346:Aquaporins; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004553:Electric Conductivity / Q000502:physiology; D005136:Eye Proteins / Q000302:isolation & purification* / Q000378:metabolism; D005614:Freeze Fracturing; D015640:Ion Channel Gating / Q000502:physiology; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D007908:Lens, Crystalline / Q000737:chemistry*; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes; D008562:Membrane Glycoproteins; D008564:Membrane Potentials / Q000502:physiology; D011189:Potassium Chloride / Q000378:metabolism; D013053:Spectrophotometry; D013395:Sucrose / Q000378:metabolism
D020346:Aquaporins; D005136:Eye Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D008562:Membrane Glycoproteins; C030328:aquaporin 0; D013395:Sucrose; D011189:Potassium Chloride