Removal of detergent from mixed micelles of egg yolk phosphatidylcholine and octyl glucoside leads to formation of unilamellar phospholipid vesicles with a diameter of about 230 nm. The same procedure applied to mixed micelles containing the transmembrane protein glycophorin A, in addition to lipid and detergent, produces vesicles of the same size with glycophorin incorporated into the bilayer. The pure lipid vesicles are highly impermeable to both anions and cations, and incorporation of up to 220 molecules of glycophorin per vesicle has little effect on permeability.

The Xenopus laevis oocyte is widely used to express exogenous channels and transporters and is well suited for functional measurements including currents, electrolyte and nonelectrolyte fluxes, water permeability and even enzymatic activity. It is difficult, however, to transform functional measurements recorded in whole oocytes into the capacity of a single channel or transporter because their number often cannot be estimated accurately. We describe here a method of estimating the number of exogenously expressed channels and transporters inserted in the plasma membrane of oocytes. The method is based on the facts that the P (protoplasmic) face in water-injected control oocytes exhibit an extremely low density of endogenous particles (212 +/- 48 particles/microns2, mean, SD) and that exogenously expressed channels and transporters increased the density of particles (up to 5,000/microns2) only on the P face. The utility and generality of the method were demonstrated by estimating the "gating charge" per particle of the Na+/glucose cotransporter (SGLT1) and a nonconducting mutant of the Shaker K+ channel proteins, and the single molecule water permeability of CHIP (Channel-like In-tramembrane Protein) and MIP (Major Intrinsic Protein). We estimated a "gating charge" of approximately 3.5 electronic charges for SGLT1 and approximately 9 for the mutant Shaker K+ channel from the ratio of Qmax to density of particles measured on the same oocytes. The "gating charges" were 3-fold larger than the "effective valences" calculated by fitting a Boltzmann equation to the same charge transfer data suggesting that the charge movement in the channel and cotransporter occur in several steps. Single molecule water permeabilities (pfs) of 1.4 x 10(-14) cm3/sec for CHIP and of 1.5 x 10(-16) cm3/sec for MIP were estimated from the ratio of the whole-oocyte water permeability (Pf) to the density of particles. Therefore, MIP is a water transporter in oocytes, albeit approximately 100-fold less effective than CHIP.

An 18-A resolution map of the 'gap junction' has been obtained by electron microscopy. The protein oligomer in the junctional membranes, the 'connexon', is a cyclinder composed of six subunits which are titled around its axis. Analysis of two different subunit configurations suggests how the connexon might regulate the passage of small molecules between cell interiors.

We have used freeze-fracture electron microscopy to examine the oligomeric structure and molecular asymmetry of integral plasma membrane proteins. Recombinant plasma membrane proteins were functionally expressed in Xenopus laevis oocytes, and the dimensions of their freezefracture particles were analyzed. To characterize the freezefracture particles, we compared the particle cross-sectional area of proteins with ␣-helical transmembrane domains (opsin, aquaporin 1, and a connexin) with their area obtained from existing maps calculated from two-dimensional crystals. We show that the cross-sectional area of the freeze-fracture particles corresponds to the area of the transmembrane domain of the protein, and that the protein cross-sectional area varies linearly with the number membrane-spanning helices. On average, each helix occupies 1.40 ؎ 0.03 nm 2 . By using this information, we examined members from three classes of plasma membrane proteins: two ion channels, the cystic fibrosis transmembrane conductance regulator and connexin 50 hemi-channel; a water channel, the major intrinsic protein (the aquaporin 0); and a cotransporter, the Na ؉ ͞glucose cotransporter. Our results suggest that the cystic fibrosis transmembrane conductance regulator is a dimer containing 25 ؎ 2 transmembrane helices, connexin 50 is a hexamer containing 24 ؎ 3 helices, the major intrinsic protein is a tetramer containing 24 ؎ 3 helices, and the Na ؉ ͞glucose cotransporter is an asymmetrical monomer containing 15 ؎ 2 helices.

Electrophysiological methods were used to assess the effect of chloride-channel blockers on the macroscopic and microscopic currents of mouse connexin50 (Cx50) and rat connexin46 (Cx46) hemichannels expressed in Xenopus laevis oocytes. Oocytes were voltage-clamped at -50 mV and hemichannel currents (ICx50 or ICx46) were activated by lowering the extracellular Ca2+ concentration ([Ca2+]o) from 5 mM to 10 microM. Ion-replacement experiments suggested that ICx50 is carried primarily (>95%) by monovalent cations (PK : PNa : PCl = 1.0 : 0.74 : 0.05). ICx50 was inhibited by 18beta-glycyrrhetinic acid (apparent Ki, 2 microM), gadolinium (3 microM), flufenamic acid (3 microM), niflumic acid (11 microM), NPPB (15 microM), diphenyl-2-carboxylate (26 microM), and octanol (177 microM). With the exception of octanol, niflumic acid, and diphenyl-2-carboxylate, the above agents also inhibited ICx46. Anthracene-9-carboxylate, furosemide, DIDS, SITS, IAA-94, and tamoxifen had no inhibitory effect on either ICx50 or ICx46. The kinetics of ICx50 inhibition were not altered at widely different [Ca2+]o (10-500 microM), suggesting that drug-hemichannel interaction does not involve the Ca2+ binding site. In excised membrane patches, application of flufenamic acid or octanol to the extracellular surface of Cx50 hemichannels reduced single channel-open probability without altering the single-channel conductance, but application to the cytoplasmic surface had no effect on the channels. We conclude that some chloride-channel blockers inhibit lens-connexin hemichannels by acting on a site accessible only from the extracellular space, and that drug-hemichannel interaction involves a high-affinity site other than the Ca2+ binding site.

Abstract. The structural organization and protein composition of lens fiber junctions isolated from adult bovine and calf lenses were studied using combined electron microscopy, immunolocalization with monoclonal and polyclonal anti-MIP and anti-MP70 (two putative gap junction-forming proteins), and freezefracture and label-fracture methods.The major intrinsic protein of lens plasma membranes (MIP) was localized in single membranes and in an extensive network of junctions having flat and undulating surface topologies. In wavy junctions, polyclonal and monoclonal anti-MIPs labeled only the cytoplasmic surface of the convex membrane of the junction. Label-fracture experiments demonstrated that the convex membrane contained MIP arranged in tetragonal arrays 6-7 nm in unit cell dimension. The apposing concave membrane of the junction displayed fracture faces without intramembrane particles or pits. Therefore, wavy junctions are asymmetric structures composed of MIP crystals abutted against particle-free membranes. In thin junctions, anti-MIP labeled the cytoplasmic surfaces of both apposing membranes with varying degrees of asymmetry. In thin junctions, MIP was found organized in both small clusters and single membranes. These small clusters also abut against particle-free apposing membranes, probably in a staggered or checkerboard pattern. Thus, the structure of thin and wavy junctions differed only in the extent of crystallization of MIP, a property that can explain why this protein can produce two different antibodylabeling patterns. A conclusion of this study is that wavy and thin junctions do not contain coaxially aligned channels, and, in these junctions, MIP is unlikely to form gap junction-like channels. We suggest MIP may behave as an intercellular adhesion protein which can also act as a volume-regulating channel to collapse the lens extracellular space.Junctions constructed of MP70 have a wider overall thickness (18-20 nm) and are abundant in the cortical regions of the lens. A monoclonal antibody raised against this protein labeled these thicker junctions on the cytoplasmic surfaces of both apposing membranes. Thick junctions also contained isolated clusters of MIP inside the plaques of MP70. The role of thick junctions in lens physiology remains to be determined.T HE lens behaves functionally as a syncytium whose fluid balance is the integrated result of membrane transport properties of anterior epithelial and fiber cells, low resistance pathways connecting the fibers and epithelial cells, and restricted extracellular space between fibers (38, 39). Because the lens displays extensive networks of close plasma membrane appositions, it has often been argued that the low resistance pathways between fibers are the result of communicating "gap" junctions. Moreover, many of these junctional structures contain a 28-kD protein called MIP, which has been regarded as the gap junction-forming protein of the lens. Numerous studies have attempted to demonstrate that the lens junctions are gap junctions and that MIP b...

In this paper we compare the water-transport properties of Aquaporin (AQP1), a known water channel, and those of the 28 kD Major Intrinsic Protein of Lens (MIP), a protein with an undefined physiological role. To make the comparison as direct as possible we measured functional properties in Xenopus laevis oocytes injected with cRNAs coding for the appropriate protein. We measured the osmotic permeability, Pf, (using rate of swelling) and the surface density of plasma membrane proteins (using freeze-fracture electron microscopy) in the same oocytes. Knowing both Pf and the number of exogenously expressed proteins in the membrane, we estimated the single-molecule permeability to be 2.8 x 10(-16) cm3/sec for MIP and 1.2 x 10(-14) cm3/sec for AQP1. As a negative control, a mutant MIP, truncated at the carboxyl-terminal, was shown by western blotting to be expressed, but this protein resulted in no increase in either water permeability or particle density. (Interestingly, the truncated protein was glycosylated, while the complete MIP transcript was not.) Water transport by MIP had a higher activation energy (approximately 7 Kcal/ mole) than water transport by AQP1 (approximately 2.5 Kcal/Mole) but a substantially lower activation energy than water flux across bare oolemma (approximately 20 Kcal/mole). Though the water-transport properties of MIP and AQP1 differ quantitatively, they are qualitatively quite similar. We conclude that MIP, like AQP1, forms water channels when expressed in oocytes. Thus water transport in the lens seems a plausible physiological role for MIP.

Presynaptic calcium channels are key regulators of neurotransmitter release. Oocyte expression studies suggest that cysteine string proteins are essential subunits or modulators of these channels. Subcellular fractionation revealed that cysteine string proteins copurify with synaptic vesicles. An average vesicle had eight protein monomers with both the amino and carboxyl termini detected on the cytoplasmic face. Thus, docked synaptic vesicles may regulate presynaptic calcium channels and neurotransmitter release.