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.

Calmodulin interacts with a C-terminus peptide from the lens membrane protein MIP26.

Lens fiber cells are coupled by communicating junctions that comprise over 50% of their appositional surfaces. The main intrinsic protein (MIP26) of lens fibers is a 28.2 kDa protein that forms large gap junction-like channels in reconstituted systems. Previously, we have shown that Ca(++)-activated calmodulin (CaM) regulates the permeability of reconstituted MIP26 channels and changes the conformation of MIP26, as measured by intrinsic fluorescence and circular dichroism spectroscopy. Examination of the MIP26 amino acid sequence has revealed a basic amphiphilic alpha-helical segment (Pep C) on the C-terminus with residue distribution similar to that found in other CaM binding proteins. To test the interaction between the amphiphilic segment and CaM, both a 20-mer peptide and trp-substituted fluorescent analog have been synthesized and purified by HPLC. Evidence from spectrofluorometric titration shows that the Pep C binds with CaM in 1:1 stoichiometry and with a kd of approximately 10 nM. Neither Ca++ nor H+ alone affects the conformation of the Pep C. However, when mixed with CaM the Pep C undergoes both a dramatic blue-shift in tryptophan fluorescence emission, indicative of strong hydrophobic interaction, and an increase in circular dichroism absorption in the alpha-helical region. Additional fluorescence blue-shift and alpha-helical content occur when Ca++ is added to the CaM:Pep C complex.

Calmodulin site at the C-terminus of the putative lens gap junction protein MIP26.

Lens fiber junctions contain cell-to-cell channels believed to be composed of a 28.2 kD protein (MIP26). Previous evidence indicates that calmodulin (CaM) is involved in the regulation of channel permeability by changing the conformation of the C terminal chain of MIP26. A study of the amino acid sequence of MIP26 has revealed an amphiphilic segment of the C-terminal chain with potential CaM-binding characteristics. To test the capacity of this chain to interact with CaM, a 20-amino acid peptide (peptide C) of appropriate sequence has been synthesized and purified by HPLC. Evidence from spectrofluorometry and circular dichroism experiments indicates that CaM interacts with and affects the conformation of peptide C, suggesting the involvement of MIP26 C-terminal chain and CaM in gating lens junction channels.

Is the C-terminal arm of lens gap junction channel protein the channel gate?

Lens gap junction channels are studied in a reconstituted system obtained by incorporating into liposomes, with or without calmodulin, the lens junction protein (MIP26) and its trypsin-cleaved product (MIP21) that lacks the C-terminal arm. Channel permeability is studied with an osmotic swelling assay. MIP26 and MIP21 liposomes swell in sucrose or polyethyleneglycol with or without Ca++ indicating the presence of large channels. Without Ca++, MIP26 and MIP21 liposomes swell in both permeants. With Ca++, MIP26-calmodulin liposomes do not swell in either permeant, indicating complete channel closure, while MIP21-calmodulin liposomes swell in sucrose but not in polyethyleneglycol. This suggests that the C-terminal arm participates in channel gating.

Lens junctions are communicating junctions.

Lens fibers are electrically coupled with each other and directly exchange dyes and metabolites. In most cells, this form of communication is mediated by gap junctions. Lens fibers lack typical gap junctions. The lens junctions, although morphologically similar to gap junctions, differ from them structurally, chemically and immunologically. Nevertheless, recent evidence suggests that indeed lens junctions are communicating junctions. The lens junction protein, MIP26, displays structural characteristics similar to other channel proteins. Once incorporated into liposomes it forms channels permeable to molecules as heavy as 1.5 kDa. Like other communicating junctions, lens junctions assume crystalline arrays and uncouple with Ca++. The liposome incorporated channels close with Ca++ and H+ in the presence of calmodulin (CaM). Partial loss of gating competency occurs after proteolytic cleavage of the C-terminal arm of MIP26. The need for a unique type of communicating junction in lens is unclear. A possibility is that this tissue has some special cell-to-cell transport requirements, in terms of size and/or charge of permeants, not shared by coupled cells of other tissues.

Functional modulation of cell coupling: evidence for a calmodulin-driven channel gate.

Much of the capacity of tissues to respond to signals as well integrated systems is due to the existence of direct cell-to-cell communication pathways. This type of communication, usually referred to as cell coupling, is based on the presence of cell-to-cell channels permeable to ions, metabolites, and regulatory compounds. The cell-to-cell channels are located at specialized regions of cell contact known as gap junctions or communicating junctions. An important aspect of cell coupling is channel permeability modulation. In recent years this feature of cell coupling has received a great deal of attention, most efforts being aimed at identifying uncoupling treatments and uncoupling agents and at determining the elements of the channel gating mechanism. This review focuses on recent studies suggesting the participation of calmodulin-like proteins in channel gating and on the application of in vitro approaches to cell coupling research-the study of permeability and gating of cell-to-cell channels incorporated into liposomes and the determination of conformational changes in isolated channel protein.

Permeability and gating of lens gap junction channels incorporated into liposomes.

The lens junction protein (MIP26), and its trypsin cleavage product (MIP21), isolated from calf fiber cells, are incorporated into liposomes and the permeability and gating of the resulting channels are studied spectrophotometrically by an osmotic swelling assay. Liposomes incorporated with either protein and loaded with Dextran T-10 swell when placed in isotonic or hypertonic KCl, sucrose or polyethyleneglycol (PEG), indicating the presence of channels permeable to molecules as large as MW 1500. In the absence of calmodulin (CaM), the permeability of either MIP26 or MIP21 channels is not altered by Ca++. On the contrary, MIP26-CaM channels reversibly close in the presence of Ca++ (10(-5)M). Preliminary experiments show channel closure with lowered pH (5.5) as well. While MIP26-CaM channels close to all the permeants tested, MIP21-CaM channels close only partially with Ca++, becoming impermeable to large probes (PEG) while remaining permeable to sucrose and KCl. This indicates that the trypsin-cleaved C-terminal arm of MIP26 is the channel gate. Evidence from spectrophotofluorometry and circular dichroism spectroscopy indicates that activated CaM changes the conformation of isolated MIP26, suggesting that channel occlusion could result from a change in protein configuration.

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

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, an in 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.

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

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 alpha-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 fluorescence emission, a 25% decrease in tryptophan fluorescence emission, and a 5% increase in the alpha-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.

Sub-threshold near-UV radiation effects on aphakic guinea pig retinas.

Monocularly aphakic guinea pigs (Cavia porcellis), prepared by removing the lens by phacoemulsification, were maintained under near-UV lighting conditions for several months. Exposure to near-UV energy was at much lower irradiance levels than that of sunlight, and was at lower than the threshold level for near-UV damage to the aphakic monkey retina as reported by Ham, et al (1). In some aphakic eyes, regenerated lens-like structures formed which scattered light appreciably. After increasing light exposure periods, the eyes of control and irradiated animals were studied histologically. Other animals were periodically examined by electroretinographic (ERG) techniques. While there was no observable histopathological damage, aphakic-UV irradiated eyes with little or no lens regrowth exhibited depressed b-waves, late time constants and altered wave forms when compared with control eyes. The results demonstrate that ambient near-UV light exposure can adversely influence retinal electrical activity in aphakic eyes at irradiance levels below threshold for morphological damage. The protective function of the lens is also supported by these findings.

The properties of mnemiopsin, a bioluminescent and light sensitive protein purified by hollow fiber techniques.

A calcium activated photoprotein, termed mnemiopsin, which emits bioluminescence upon the addition of calcium ion, has been isolated from the Ctenophore, Memiopsis leidyi, and purified by hollow fiber techniques. The system is similar to aequorin, from the jellyfish Aequorea, except that mnemiopsin can be light-inactivated. Separation of mnemiopsin from the dilute and large volume animal homogenate proved difficult with conventional biochemical techniques. A continuous flow process utilizing large surface area hollow fibers for filtration, concentration, and dialysis was developed which may also be applicable to the purification of other proteins. The resulting mnemiopsin concentrate, after further purification, was judged to be about 90% pure by its gel electrophoretic profile. Estimates by molecular sieve chromatography and SDS gel electrophoresis gave a molecular weight of about 23,000 daltons. A calcium specificity for triggering light emission was studied by comparison of triggering with a variety of cations and anions and by investigating the effects of calcium ionophores and antagonists. The activity of mnemiospin was characterized with respect to pH, temperature and ionic strength. The stability of mnemiopsin activity after exposure to proteases, denaturants, protein group specific reagents, detergents, elevated temperatures and light was determined. Some years ago our laboratory reported that the bioluminescence reaction in the ctenophores which had long eluded definition involved a calcium activated photoprotein similar in many respects to that found in other coelenterates, notably Aequorea. We found, moreover, that the systems differed in that the bioluminescent activity of the isolated protein was lost following exposure to light. The purification and characterization of this biochemical system was undertaken both in our laboratory and by Ward and Seliger. These latter reports provide a detailed and firm foundation for the understanding of the components and mechanisms involved. While many of our results are in agreement with theirs, our approaches, inquiries, and results differed in several significant ways, the description of which forms the basis for this report. In particular, we took a different approach in the purification of the Mnemiopsis photoprotein which in itself is rather a formidable task. The technique was successful and may point the way to other applications where large volume dilute solutions prove cumbersome. Secondly, our study of the effects of salts, proteases, detergents, and other agents indicate that the protein, though sensitive to calcium and visible light inactivation, is relatively resistant to some agents which commonly inactivate proteins.