Lens cell-to-cell channel protein: I. Self-assembly into liposomes and permeability regulation by calmodulin

Summary Lens fibers are coupled by communicating junctions which contain a 28-kDalton protein (MIP26) believed to be the main component of the cell-to-cell channel. To study the permeability properties and regulation of these channels, anin vitro system has been developed in which MIP26 isolated from calf lens is incorporated into liposomes and the resulting channels are studied spectrophotometrically by a swelling assay. Liposome vesicles were prepared using a sonication/resuspension method. Incorporation efficiency was monitored by freeze-fracture. Vesicles were resuspended in 6% Dextran T-10. Assay buffer was identical, except for isotonic substitution of sucrose for T-10. MIP26-incorporated (but not control) vesicles swell under isotonic conditions indicating sucrose entry (via channels) followed by water to maintain osmotic balance. In the absence of calmodulin, calcium ion has no effect on channel permeability. On the contrary, vesicles prepared with equimolar amounts of MIP26 and CaM do not swell in the presence of calcium ion, indicating that the channels can be closed. Addition of EGTA to these vesicles reinitiates swelling—evidence that the channel gating mechanism is reversible. Magnesium ion has no effect on either type of vesicle.     

Functional reconstitution of lens gap junction proteins into proteoliposomees

Summary Membranes rich in junction complexes were prepared from bovine lens, and the fragments of the membranes were reconstituted into proteoliposomes with a large excess of phosphatidylcholine and dicetylphosphate. The osmotic swelling behavior of these liposomes showed that the lens junction membranes contributed protein components that produced channels with a nominal diameter of 1.4 nm. Most preparations of lens junctions produced rates of osmotic swelling much slower than those found in proteoliposomes containing equivalent amounts ofEscherichia coli porin, and we discuss several possible explanations for this observation.     

Reconstitution of channels from preparations enriched in lens gap junction protein MP70

Summary Detergent-solubilized ovine lens membrane proteins, enriched in the 70-kDa gap junction component (MP70), were reconstituted into planar lipid bilayers and analyzed for channel activities. Three distinct activities were found. Those showing conductance steps of 290 pS (symmetrical 150-mM KCl solutions) had properties similar to those reported earlier for MIP26 (Ehring, G.R., Zampighi, G., Horwitz, J., Bok, D., Hall, J.E. 1990. J. Gen. Physiol. 96:631–664.) of which minor amounts were normally present in the detergent-solubilized preparations. Two novel channel activities had unitary conductances of 90 and 45 pS, were halothane sensitive and did not discriminate between sodium and potassium ions. The 90-pS channel was asymmetrically voltage dependent, and its properties would be consistent with the expected properties of junctional hemichannels.This work was supported by grants from the Health Research Council of New Zealand, from the New Zealand Lottery Grants Board and from the New Zealand Vice Chancellor's Committee Fund.     

Polypeptide neurotoxins modify gating and apparent single-channel conductance of veratridine-activated sodium channels in planar lipid bilayers

Summary The effects of scorpion and sea anemone polypeptide toxins on partially purified veratridine (VER)-activated Na channels from rat brain were studied at the single-channel level in planar lipid bilayers. The probability of the VER-activated channel being open (P _o ) increased with depolarization;P _o was 0.5 at –40 to –50 mV. Saxitoxin (STX) blocked VER-activated channels with an apparent dissociation constant of about 1nm at –45 mV. The apparent single-channel conductance was approximately 9 pS, similar to that seen in VER-activated Na channels from skeletal muscle transverse tubules. Addition of sea anemone or scorpion polypeptide toxins to VER-activated Na channels resulted in a 19% increase in apparent single-channel conductance and a hyperpolarizing shift in theP _o vs. V _m relation such that the channels were more likely to be open at potentials <40 mV. These effects of the polypeptide toxins on the single-channel properties of VER-activated Na channels may account for the previously described potentiation of VER action by polypeptide toxins.     

Single-membrane and cell-to-cell permeability properties of dissociated embryonic chick lens cells

Ion channels are believed to play an important role in the maintenance of lens transparency. In order to ascribe junctional and nonjunctional permeability properties to specific lens cell types, embryonic chick lenses were enzymatically dissociated into cell clusters, cell pairs and single cells, and both cell-to-cell and single-membrane permeability properties were characterized with the patch-clamp technique. Double patch-clamp experiments and single patch-clamp experiments with Lucifer yellow in the pipette demonstrated that the cells in the dissociated preparation were well coupled, the average conductance between pairs being 42 +/- 27 nS. Double patch-clamp experiments also revealed single cell-to-cell channel events with a predominant unitary conductance of 286 +/- 38 pS. Whole-cell measurements of surface membrane conductance indicate heterogeneity within the population of dissociated embryonic chick lens cells: 63% of the cells have a voltage-independent leak current, 14% of the cells have a potassium-selective inward-rectifier current, and 23% of the cells have a current which turns off with positive voltage on a time scale on the order of seconds. The time constant for this turnoff is voltage dependent.     

Phosphorylation modulates the voltage dependence of channels reconstituted from the major intrinsic protein of lens fiber membranes

Summary Major intrinsic polypeptide (MIP), a 28-kDa protein isolated from lens fiber cell membranes, forms large, nonselective channels when reconstituted into lipid bilayers. MIP channels are regulated by voltage, such that these channels close when the potential across the membrane is greater than 30 mV. We have investigated the modulation of the voltage-dependent closure of MIP channels by phosphorylation. In this report, we describe the isolation of two isomers of MIP from lens fiber cell membranes. These isomers differ by a single phosphate at a protein kinase A phosphorylation site. The phosphorylated isomer produces channels that close in response to applied voltages when reconstituted into bilayers. The nonphosphorylated isomer produces voltage-independent hannels. Direct phosphorylation with protein kinase A converts voltage-independent channels to voltage-dependent channels in situ. Analyses of macroscopic and single channel currents suggest that phosphorylation increases the voltage-dependent closure of MIP channels by increasing closed channel lifetimes and the rate of channel closure following the application of voltage.The authors gratefully acknowledge the gift of the monoclonal antibody to MIP from Drs. David Paul and Dan Goodenough. We thank Dr. Irwin Levitan for the kind gift of purified protein kinase A catalytic subunit. We also thank Ms. Mary Hawley for invaluable technical support and Mr. Paul Ross for help in generating Fig. 10. This work was supported, in part, by NIH grants EY04110 and EY05661 and a NEI postdoctoral fellowship to GRE.     

Gating processes of channels induced by colicin A,its C-terminal fragment and colicin E1 in planar lipid bilayers

The dependence on pH and membrane potential of the pore formed by colicin A and its C-terminal 20 kDa fragment has been measured using planar lipid bilayers. The single channel conductance of the pore formed by both colicin A and the fragment increases with pH with an apparent pK of 6.0. At pH 5.0 the gating by membrane potential of the channels formed by either colicin A or its fragment is identical. At the same pH, quite similar pore properties were found when using the related bacteriocin, colicin E₁. In agreement with previous studies, these data indicate that the protein structure containing the lumen of the pore resides in the 20 kDa C-terminal part of the colicin A and favours the recently proposed model, based on protein sequence analysis, which proposes that colicin A, E₁ and I_B C-terminal domains are folded in the same three-dimensional structure. However, it is also shown that colicin A and not its C-terminal fragment undergoes a pH dependent transition between an acidic and a basic form of the pore with an apparent pK of 5.3. The two forms of the pore differ by their gating charge but not by the channel size. These results suggest that there is a pH dependent association between the C-terminal domain carrying the lumen of the pore and another domain of the molecule which affect the pore sensitivity to membrane potential.     

Lens cell-to-cel channel protein: II. Conformational change in the presence of calmodulin

Summary Lens fibers are coupled by communicating junctions, clusters of cell-to-cell channels composed of a 28-kD intrinsic membrane protein (MIP26). Evidence suggests that these and other cell-to-cell channels may close as a result of protein conformational change induced by activated calmodulin. To test the validity of this hypothesis, we have measured the intrinsic fluorescence emission and far-ultraviolet circular dichroism of the isolated components MIP26, calmodulin, and the MIP26-calmodulin complex, both in the absence and presence of Ca⁺⁺, an uncoupling agent. MIP26 shows no change in either, fluorescence emission (primarily tryptophan and a measure of aromatic constitutivity) or in its circular dichroism spectrum. Calmodulin exhibits a 32% increase in fluorescence emission intensity with constant emission wavelength, entirely tyrosine, and a 44% increase in -helicity, changes previously described. The MIP26-calmodulin complex, on the other hand, displays fluorescence emission and circular dichroism spectra which are slightly different from the sum of the two single components, but shows marked differences in both spectra upon Ca⁺⁺ addition. This indicates a change in conformation in one or both of the two components. Spectral changes include a 5-nm blue-shift, a 50% increase in tyrosine fluorescene emission, a 25% decrease in tryptophan fluorescence emission, and a 5% increase in the -helicity of the complex. These changes also occur about an isosbestic point and are fully reversible. These data provide additional evidence that activated calmodulin may modulate gating of cell-to-cell channels by affecting channel protein.     

Phosphorylation of MP26, a lens junction protein,is enhanced by activators of protein kinase C

Summary MP26, a protein thought to form gap junctional channels in the lens, and other lens proteins were phosphorylated under conditions that activate protein kinase C. Phosphorylation was detected both in lens fiber cell fragments in an in vivo labeling procedure with³²P-phosphate and in cell homogenates with³²P-ATP. In these experiments, both calcium and 12-O-tetradecanoylphorbol 13-acetate (TPA) were necessary for maximal phosphorylation of MP26. Calcium stimulated the phosphorylation of MP26 approximately fourfold and TPA with calcium led to a sevenfold increase. If TPA was present, 1 m calcium was sufficient for maximal labeling. Phosphoamino acid analysis demonstrated approximately 85% phosphoserine, 15% phosphothreonine, and no phosphotyrosine when MP26 was phosphorylated in lens homogenates in the presence of TPA and calcium and then electrophoretically purified. Phosphorylation occurred near the cytoplasmic, C-terminal of MP26. The possible involvement of other kinases was also examined. The Walsh inhibitor, which affects cAMP-dependent protein kinases, had no influence on the TPA-mediated increase in phosphorylation. In studies with isolated membranes and added kinases, MP26 was also found to not be a substrate for calcium/calmodulindependent protein kinase II. Thus, protein kinase C may have phosphorylated MP26 in a direct manner.     

Properties of single- and double-barreled Cl channels of shark rectal gland in planar bilayers

Chloride channels from the apical plasma membrane fraction of rectal gland of Squalus acanthias were characterized by incorporation into planar bilayers in the presence of cAMP-PK/ATP. In a total of 80 bilayer preparations, 21 Cl-selective channels were observed as single channels and 13 as pairs. This was a significantly greater number of double Cl channels than expected from a binomial distribution. The double Cl channels were divided into two groups based on kinetic and voltage-dependent behavior. One group had properties identical to the single channels (gb1) while the other was consistent with a double-barreled channel (gb2) with coordinated activity between proto-channels. The single-channel slope conductances of gb1 and gb2 from -60 to +20 mV with a 250/70 mM KCl gradient were 41 and 75 pS, respectively. With symmetrical 250 mM KCl, the I-V relation of gb1 showed outward rectification with 47.8 +/- 6.6 pS at cis negative potentials and 68.9 +/- 6.1 pS at cis positive potentials. gb1 was open from 70 to 95% at all electrochemical potentials from -80 to +40 mV. gb2 was steeply voltage dependent between -80 and -20 mV. Both gb1 and gb2 were insensitive to Ca (from 100 nm to 1 microM), blocked by 0.1 mM DIDS and highly selective for chloride. These data suggest that double-barreled Cl channels are related to the family of small, outwardly rectifying Cl channels of epithelial membranes.     

Arrangement of MP26 in lens junctional membranes: Analysis with proteases and antibodies

Summary The major membrane protein of the bovine lens fiber cell is a 26-kilodalton (kD) protein (MP26), which appears to be a component of the extensive junctional specializations found in these cells. To examine the arrangement of MP26 within the junctional membranes, various proteases were incubated with fiber cell membranes that had been isolated with or without urea and/or detergents. These membranes were analyzed with electron microscopy and SDS-PAGE to determine whether the junctional specializations or the proteins were altered by proteolysis. Microscopy revealed no obvious structural changes. Electrophoresis showed that chymotrypsin, papain, and trypsin degraded MP26 to 21–22 kD species. A variety of protease treatments, including overnight digestions, failed to generate additional proteolysis. Regions on MP26 which were sensitive to these three proteases overlapped. Smaller peptides were cleaved from MP26 with V8 protease and carboxypptidases A and B. Protein domains cleaved by these proteases also overlapped with regions sensitive to chymotrypsin, papain, and trypsin. Specific inhibition of the carboxypeptidases suggested that cleavage obtained with these preparations was not likely due to contaminating endoproteases. Since antibodies are not thought to readily penetrate the 2–3 nm extracellular gap in the fiber cell junctions, antibodies to MP26 were used to analyze the location of the protease-sensitive domains. Antisera were applied to control (26 kD) and proteolyzed (22 kD) membranes, with binding being evaluated by means of ELISA reactions on intact membranes. Antibody labeling was also done following SDS-PAGE and transfer to derivatized paper. Both assays showed a significant decrease in binding following proteolysis, with the 22 kD product showing no reaction with the anti-MP26 sera. These investigations suggest that MP26 is arranged with approximately fourfifths of the primary sequence “protected” by the lipid bilayer and the narrow extracellular gap. One-fifth of the molecule, including the C-terminus, appears to be exposed on the cytoplasmic side of the membrane.     

Confocal fluorescence microscopy study of interaction between lens MIP26/AQP0 and crystallins in living cells

MIP26/AQP0 is the major lens fiber membrane protein and has been reported to interact with many other lens components including crystallins, lipid, and cytoskeletal proteins. Regarding crystallins, many previous reports indicate that MIP26/AQP0 interacts with either only alpha-crystallin or some specific gamma-crystallins. Considering the possibly important role of MIP26/AQP0 in the reduction of light scattering in the lenses, we have further investigated its interaction with crystallins using confocal fluorescence resonance energy transfer (FRET) microscopy. Specifically, we used MIP26 tagged with a green fluorescence protein (GFP) as a donor and a crystallin (alphaA-, alphaB-, betaB2-, or gammaC-crystallin) tagged with a red fluorescence protein (RFP) as an acceptor. The two plasmids were cotransfected to HeLa cells. After culture, laser scattering microscopy images were taken in each of the three channels: GFP, RFP, and FRET. The net FRET images were then obtained by removing the contribution of spectral bleed-through. The pixels of net FRET were normalized with those of GFP. The results show the presence of measurable interactions between MIP26 and all crystallins, with the extent of interactions decreasing from alphaA- and alphaB-crystallin to betaB2- and gammaC-crystallin. Competitive interaction study using untagged alphaA-crystallin shows decreased net FRET, indicating specificity of the interactions between MIP26 and alphaA-crystallin. We conclude that all crystallins interact with MIP26, the physiological significance of which may be a reduction in the difference of refractive index between membrane and cytoplasm.     

Phloretin and phloretin analogs: Mode of action in planar lipid bilayers and monolayers

Summary Phloretin and other neutral phloretin-like molecules are able to decrease the electrostatic potential within neutral lipid bilayers and monolayers. The relationship between the change in the dipole potential and the aqueous concentration of the molecule is well described by a Langmuir isotherm. From the Langmuir isotherm, the apparent dissociation constants (K _D ^A ) and the maximum dipole potential change ( _max) are obtained for the different phloretin-like molecules tested. Considering the phloretin analogs as derivatives of acetophenone containing two kinds of substituents, one on the benzene ring and another on the carbon chain, it is found that (a)K _D ^A is related to the hydrophobicity of the compound and is also a function of the position of the hydroxyl substituent in the ring; (b) from the dependence ofK _D ^A on the length of the acyl chain, it is estimated that the free-energy change is 650 cal/mole CH₂; (c) _max is not a simple function of the dipole moment of the molecule but depends on the substituent on the carbon chain and on the position and number of hydroxyl groups on the benzene ring; (d) phloretin adsorption parameters are a function of membrane lipid composition. The results are discussed in terms of the effect of these compounds on chloride transport in red blood cells.     

Pore formation by complement in the outer membrane of Gram-negative bacteria studied with asymmetric planar lipopolysaccharide/phospholipid bilayers

Summary The interaction of complement with an asymmetric planar lipopolysaccharide/phospholipid bilayer system as a model for the lipid matrix of the outer membrane of Gram-negative bacteria has been studied. The addition of whole human serum to the aqueous solution at the lipopolysaccharide side of the asymmetric membrane resulted in a rapid increase of the bilayer conductance in discrete steps, indicating the formation of transmembrane pores, which were not observed in the case of pure phospholipid membranes. The amplitudes of the discrete conductance steps varied over a range of more than one order of magnitude. The mean single step conductance was (0.39±0.24) nS for a subphase containing (inmm): 100 KCl, 5 MgCl₂ and 5 HEPES buffer. The steps were grouped into bursts of typically 9±3 events per burst and the conductance change within one burst was (8.25±4.00) nS.The pore-forming activity of serum at the asymmetric membrane system was independent of the presence of specific antibodies against the lipopolysaccharide but was dependent on calcium ions. Furthermore, the pore-forming activity required complement component C9.A model for the mode of pore formation by complement is proposed: The complement pore is generated in discrete steps by insertion of C9 monomers into the membrane and their irreversible aggregation to water-filled channels with a diameter of approximately 7 nm assuming a circular geometry.     

Template-assembled melittin: structural and functional characterization of a designed, synthetic channel-forming protein.

Template-assembled proteins (TASPs) comprising 4 peptide blocks, each of either the natural melittin sequence (melittin-TASP) or of a truncated melittin sequence (amino acids 6-26, melittin6-26-TASP), C-terminally linked to a (linear or cyclic) 10-amino acid template were synthesized and characterized, structurally by CD, by fluorescence spectroscopy, and by monolayer experiments, and functionally, by electrical conductance measurements on planar bilayers and release experiments on dye-loaded vesicles. Melittin-TASP and the truncated analogue preferentially adopt alpha-helical structures in methanol (56% and 52%, respectively) as in lipid membranes. Unlike in methanol, the melittin-TASP self-aggregates in water. On an air-water interface, the differently sized molecules can be self-assembled and compressed to a compact structure with a molecular area of around 600 A2, compatible with a 4-helix bundle preferentially oriented perpendicular to the interface. The proteins reveal a strong affinity for lipid membranes. A partition coefficient of 1.5 x 10(9) M-1 was evaluated from changes of the Trp fluorescence spectra of the TASP in water and in the lipid bilayer. In planar lipid bilayers, TASP molecules are able to form defined ion channels, exhibiting a small single-channel conductance of 7 pS (in 1 M NaCl). With increasing protein concentration in the lipid bilayer, additional, larger conductance states of up to 1 nS were observed. These states are likely to be formed by aggregated TASP structures as inferred from a strongly voltage-dependent channel activity on membranes of large area. In this respect, melittin-TASP reveals channel features of the native peptide, but with a considerably lower variation in the size of the channel states. Compared to the free peptide, template-assembled melittin has a much higher membrane activity: it is about 100 times more effective in channel formation and 20 times more effective in releasing dye molecules from lipid vesicles. This demonstrates that the lytic properties are not solely related to channel formation.     

Reconstitution of a calcium-activated potassium channel in basolateral membranes of rabbit colonocytes into planar lipid bilayers

Summary A highly enriched preparation of basolateral membrane vesicles was isolated from rabbit distal colon surface epithelial cells employing the method described by Wiener, Turnheim and van Os (Weiner, H., Turnheim, K., van Os, C.H. (1989)J. Membrane Biol.110:147–162) and incorporated into planar lipid bilayers. With very few exceptions, the channel activity observed was that of a high conductance, Ca²⁺-activated K⁺ channel. This channel is highly selective for K⁺ over Na⁺ and Cl^–, displays voltage-gating similar to maxi K(Ca) channels found in other cell membranes, and kinetic analyses are consistent with the notion that K⁺ diffusion through the channel involves either the binding of a single K⁺ ion to a site within the channel or single-filling (multi-ion occupancy). Channel activity is inhibited by the venom from the scorpionLeiurus quinquestriatus, Ba²⁺, quinine, and trifluoperazine. The possible role of this channel in the function of these cells is discussed.     

Molecular determinants of electrical rectification of single channel conductance in gap junctions formed by connexins 26 and 32.

The fully open state of heterotypic gap junction channels formed by pairing cells expressing connexin 32 (Cx32) with those expressing connexin 26 (Cx26) rectifies in a way that cannot be predicted from the current-voltage (I-V) relation of either homotypic channel. Using a molecular genetic analysis, we demonstrate that charged amino acids positioned in the amino terminus (M1 and D2) and first extracellular loop (E42) are major determinants of the current-voltage relation of the fully open state of homotypic and heterotypic channels formed by Cx26 and Cx32. The observed I-V relations of wild-type and mutant channels were closely approximated by those obtained with the electrodiffusive model of Chen and Eisenberg (Chen, D., and R. Eisenberg. 1993. Biophys. J. 64:1405-1421), which solves the Poisson-Nernst-Plank equations in one dimension using charge distribution models inferred from the molecular analyses. The rectification of the Cx32/Cx26 heterotypic channel results from the asymmetry in the number and position of charged residues. The model required the incorporation of a partial charge located near the channel surface to approximate the linear I-V relation observed for the Cx32*Cx26E1 homotypic channel. The best candidate amino acid providing this partial charge is the conserved tryptophan residue (W3). Incorporation of the partial charge of residue W3 and the negative charge of the Cx32E41 residue into the charge profile used in the Poisson-Nernst-Plank model of homotypic Cx32 and heterotypic Cx26/Cx32 channels resulted in I-V relations that closely resembled the observed I-V relations of these channels. We further demonstrate that some channel substates rectify. We suggest that the conformational changes associated with transjunctional voltage (V(j))-dependent gating to these substates involves a narrowing of the cytoplasmic entry of the channel that increases the electrostatic effect of charges in the amino terminus. The rectification that is observed in the Cx32/Cx26 heterotypic channel is similar although less steep than that reported for some rectifying electrical synapses. We propose that a similar electrostatic mechanism, which results in rectification through the open and substates of heterotypic channels, is sufficient to explain the properties of steeply rectifying electrical synapses.     

Basolateral membrane K permselectivity and regulation in bullfrog cornea epithelium

Summary Ion channels permeable to barium and calcium were reconstituted from theAplysia nervous system into phospholipid bilayers formed on the tips of patch electrodes. With asymmetrical concentrations of barium or calcium on the two sides of the bilayer, the single-channel currents reversed at the calculated barium or calcium reversal potentials, indicating that the channels were cation selective. Channels with conductances of 10, 25 and 36 pS were routinely observed. Calcium and barium were equally effective as charge carriers for the 36-pS channel, whereas magnesium was at least fifteenfold less effective. The gating of all three channels was independent of the voltage across the bilayer, but was affected by the dihydropyridine calcium channel agonist Bay K 8644 (Bay K). In the presence of Bay K but not in its absence, long discrete gating events were routinely observed, suggesting that the dihydropyridine increased the probability of long open states as it does for calcium channels in other systems.Bilayers invariably contained more than a single channel (or conductance state). This was observed even when theAplysia nervous system membranes were prepared in the presence of cytoskeleton disrupting agents, or when the membrane proteins were diluted extensively with exogenous phospholipid. Furthermore, transitions between conductance levels were observed with high frequency. These findings, together with the fact that all of the conductance states share certain properties including voltage-independence and sensitivity to Bay K, suggest that the apparent multiple channel types may in fact represent subconductance states of a single ion channel.