Membrane insertion and assembly of ductin: a polytopic channel with dual orientations.

Ductin is a highly conserved and polytopic transmembrane protein which is the subunit c component of the vacuolar H(+)-ATPase (V-ATPase) and a component of a connexon channel of gap junctions. Previous studies have suggested that ductin in the V-ATPase has the opposite orientation of ductin in a connexon. Using an in vitro translation system coupled to microsomes derived from the endoplasmic reticulum, we show that ductin is co-translationally inserted into the membrane bilayer, suggesting a dependency on the signal recognition particle for synthesis. By attaching a C-terminal polypeptide derived from beta-lactamase and by using cysteine replacement coupled to chemical labelling, we show that ductin is inserted into the microsomal membrane in both orientations in similar proportions. In contrast, squid rhodopsin appears to be inserted in a single orientation. Changing conserved charged residues at the N-terminus of ductin does not affect the ratio of the two orientations. Once in the microsomal membrane, ductin assembles into an oligomeric complex which contains a pore accessible to a water-soluble probe, reminiscent of the ductin complex found in the V-ATPase and a connexon.     

Structure of the Ductin Channel

Gap junctions appear to be essential components of metazoan animals providing a means of direct means of communication between neighboring cells. They are sieve-like structures which allow cell–cell movement of cytosolic solutes below 1000 MW. The major role of gap junctions would appear to be homeostatic giving rise to groups of cells which act as functional units. Ductin is the major core component of gap junctions and recent structural data shows it to be a four alpha-helical bundle which fits particularly well into a low resolution model of the gap junction channel. Ductin is also the main membrane component of the vacuolar H₊-ATPase that is found in all eukaryotes and it seems likely that the gap junction channel first evolved as a housing for the rotating spindle of these proton pumps. Because ductin protrudes little from the membrane, other proteins are required to bring cell surfaces close enough together to form gap junctions. Such proteins may include connexins, a large family of proteins found in vertebrates.     

Disposition and Orientation of Ductin (DCCD-Reactive Vacuolar H-ATPase Subunit) in Mammalian Membrane Complexes

The disposition and orientation of mouse ductin (the subunit c of the vacuolar H⁺-ATPase) in gap junctions has been examined. Like the Nephrops norvegicus (arthropod) form, mouse ductin in the intact junctional structure is resistant to high levels of nonspecific proteinase, suggesting that it is for the most part buried in the bilayer. Antisera to an octapeptide near the N-terminus cross-react with ductins in gap junction preparations from four different mouse tissues, from chicken and Xenopus laevis liver, and from N. norvegicus hepatopancreas. The antisera and antibodies, affinity purified against the octapeptide, agglutinate isolated gap junctions, suggesting that the N-terminus is located on the exposed surface, equivalent to the cytoplasmic face of an intercellular gap junction. The antibodies also block dye coupling when injected into cells in culture, confirming the cytoplasmic location of the epitope. The lipophylic reagent dicylohexyl carbodiimide (DCCD), which targets carboxyl groups within the membrane and selectively reacts with ductin in N. norvegicus gap junction preparations, rapidly inhibits junctional communication. Bafilomycin A₁, which inhibits V-ATPase and stops vacuolar acidification, does not affect dye coupling, showing that the inhibition seen with antibodies and DCCD is not an indirect consequence of their action on the ductin of V-ATPase. Consistent with this interpretation the anti-peptide antibodies do not bind to intact chromaffin granules or inhibit their V-ATPase activity, but do bind to osmotically disrupted granule membrane. This suggests that ductin has an orientation (N-terminus pointing away from the cytoplasm) in the vacuolar membrane opposite to that in the gap junction membrane.     

Photoaffinity labelling of a cytokinin-binding integral membrane protein in plant mitochondria

Two target polypeptides were detected by photoaffinity labelling of purified mung bean mitochondria using tritiated 2-azido-N⁶-benzylaminopurine. SDS-PAGE and fluorography of total mitochondrial proteins after the photoaffinity reaction showed a labelled 32 kDa polypeptide (intensely labelled) and a 57 kDa polypeptide (less intensely labelled). The latter was assumed to be the and/or subunit of F₁ATPase since it was the most abundant polpeptide in gels stained with Coomassie Blue. Partial purification of F₁ATPase demonstrated that the 32 kDa polypeptide was not a component of the ATPase complex. Fractionation experiments showed that the 32 kDa protein was integrally associated with mitochondrial membranes and could be enriched by simple washing and detergent extraction procedures.     

ATPaseTb2, a Unique Membrane-bound FoF1-ATPase Component,Is Essential in Bloodstream and Dyskinetoplastic Trypanosomes

In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt) membrane potential (Δψ_m) is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential F_oF₁-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψ_m that manifests as a decreased growth phenotype, indicating that the F_oF₁-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) and thus subunit a, an essential component of the proton pore in the membrane F_o-moiety. These Dk cells generate the Δψ_m by combining the hydrolytic activity of the matrix-facing F₁-ATPase and the electrogenic exchange of ATP⁴- for ADP³- by the ATP/ADP carrier (AAC). Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric F_oF₁-ATPase complexes were identified. In fact, the immunoprecipitation of a F₁-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψ_m of Dk cells. Thus, even in the absence of subunit a, a portion of the F_oF₁-ATPase is assembled in Dk cells.     

Ductin, a component of the V-ATPase, is developmentally regulated in Heliothis virescens midgut, and anti-ductin antibodies label lateral membranes

We previously cloned from Heliothis virescens a 16-kDa protein that is homologous to other ductin sequences. We also reported its immunolocalization with a specific affinity-purified anti-peptide antibody in the midgut and Malpighian tubule of feeding larvae, and concluded that the cloned proteolipid encodes the V-ATPase proton-transporting subunit c from the V₀ sector. We now present the immunolocalization of this protein in the midgut during the L₄-L₅ larval molt and early post-ecdysis into the fifth instar in H. virescens. The results show that the spatial expression of the 16-kDa protein is developmentally regulated. Labeling by anti-peptide antibody varies during the molt in the midgut goblet cell apical plasma membrane and the goblet cell apical valve. Epifluorescence and confocal microscopy revealed strong anti-ductin labeling in areas of cell-to-cell contact during the molt, and during early post-ecdysis into the fifth larval instar. The characteristic labeling pattern observed in areas of cell-to-cell contact is consistent with the claimed involvement of ductins in gap junctions. Conclusive evidence for the presence of the 16-kDa protein in areas of cell-to-cell contact in the midgut of feeding larvae is, however, lacking. V-ATPase regulation during the molt was also investigated by simultaneous immunohistochemistry with an anti-B subunit antiserum, a probe for the V₁ sector. Received: 2 April 1996 / Accepted: 14 November 1996     

Potential role of subunit c of F0F1-ATPase and subunit c of storage body in the mitochondrial permeability transition. Effect of the phosphorylation status of subunit c on pore opening

Phosphorylated and non-phosphorylated forms of the F₀F₁-ATPase subunit c from rat liver mitochondria (RLM) were purified and their effect on the opening of the permeability transition pore (mPTP) was investigated. Addition of dephosphorylated subunit c to RLM induced mitochondrial swelling, decreased the membrane potential and reduced the Ca²⁺ uptake capacity, which was prevented by cyclosporin A. The same effect was observed in the presence of storage subunit c purified from livers of sheep affected with ceroid lipofuscinosis. In black-lipid bilayer membranes subunit c increased the conductance due to formation of single channels with fast and slow kinetics. The dephosphorylated subunit c formed channels with slow kinetics, i.e. the open state being of significantly longer duration than in the case of channels formed by the phosphorylated form that had short life spans and fast kinetics. The channels formed were cation-selective more so with the phosphorylated form. Subunit c of rat liver mitochondria was able to bind Ca²⁺. Collectively, the data allowed us to suppose that subunit c F₀F₁-ATPase might be a structural/regulatory component of mPTP exerting its role in dependence on phosphorylation status.     

Diet-induced obesity impairs endothelium-derived hyperpolarization via altered potassium channel signaling mechanisms

Background

The vascular endothelium plays a critical role in the control of blood flow. Altered endothelium-mediated vasodilator and vasoconstrictor mechanisms underlie key aspects of cardiovascular disease, including those in obesity. Whilst the mechanism of nitric oxide (NO)-mediated vasodilation has been extensively studied in obesity, little is known about the impact of obesity on vasodilation to the endothelium-derived hyperpolarization (EDH) mechanism; which predominates in smaller resistance vessels and is characterized in this study.

Methodology/Principal Findings

Membrane potential, vessel diameter and luminal pressure were recorded in 4^th order mesenteric arteries with pressure-induced myogenic tone, in control and diet-induced obese rats. Obesity, reflecting that of human dietary etiology, was induced with a cafeteria-style diet (∼30 kJ, fat) over 16–20 weeks. Age and sexed matched controls received standard chow (∼12 kJ, fat). Channel protein distribution, expression and vessel morphology were determined using immunohistochemistry, Western blotting and ultrastructural techniques. In control and obese rat vessels, acetylcholine-mediated EDH was abolished by small and intermediate conductance calcium-activated potassium channel (SK_Ca/IK_Ca) inhibition; with such activity being impaired in obesity. SK_Ca-IK_Ca activation with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) and 1-ethyl-2-benzimidazolinone (1-EBIO), respectively, hyperpolarized and relaxed vessels from control and obese rats. IK_Ca-mediated EDH contribution was increased in obesity, and associated with altered IK_Ca distribution and elevated expression. In contrast, the SK_Ca-dependent-EDH component was reduced in obesity. Inward-rectifying potassium channel (K_ir) and Na⁺/K⁺-ATPase inhibition by barium/ouabain, respectively, attenuated and abolished EDH in arteries from control and obese rats, respectively; reflecting differential K_ir expression and distribution. Although changes in medial properties occurred, obesity had no effect on myoendothelial gap junction density.

Conclusion/Significance

In obese rats, vasodilation to EDH is impaired due to changes in the underlying potassium channel signaling mechanisms. Whilst myoendothelial gap junction density is unchanged in arteries of obese compared to control, increased IK_Ca and Na⁺/K⁺-ATPase, and decreased K_ir underlie changes in the EDH mechanism.     

Insertion of a “chloroplast-like” regulatory segment responsible for thiol modulation into γ-subunit of F0F1-ATPase of the cyanobacterium Synechocystis 6803 by mutagenesis of atpC

A regulatory sequence in the γ subunit of the F₀F₁-ATPase complex of higher plant chloroplasts, responsible for so-called thiol modulation, is absent in the corresponding polypeptides of the cyanobacterial complexes analysed so far. We have modified the atpC gene encoding this γ subunit in Synechocystis 6803 by site-directed mutagenesis. A segment was introduced coding for nine additional amino acids, including the two functional cysteines, which constitutes the sequence of the respective element in the chloroplast γ subunit. The growth rate as well as the rate of photosynthesis of the transformant was comparable to that of the wild-type, but the transitory increase in respiration observed immediately after a period of illumination was significantly lower in the mutant than in the wild-type. The F₁ subcomplex solubilized from thylakoid membranes of both the wild-type and the transformant can be activated by trypsin to yield Ca²⁺-dependent ATPase activity, but only the F₁ from the transformant can be activated by the thiol reagent dithiothreitol.     

The muscle ryanodine receptor and its intrinsic Ca2+ channel activity

In skeletal and cardiac muscle, contraction is initiated by the rapid release of Ca²⁺ ions from the intracellular membrane system, sarcoplasmic reticulum. Rapid-mixing vesicle ion flux and planar lipid bilayer-single-channel measurements have shown that Ca²⁺ release is mediated by a high-conductance, ligand-gated Ca²⁺ channel. Using the Ca²⁺ release-specific probe ryanodine, a 30 S protein complex composed of four polypeptides ofM _r 400,000 has been isolated. Reconstitution of the purified skeletal and cardiac muscle 30 S complexes into planar lipid bilayers induced single Ca²⁺ channel currents with conductance and gating kinetics similar to those of native Ca²⁺ release channels. Electron microscopy revealed structural similarity with the protein bridges (feet) that span the transverse-tubule-sarcoplasmic reticulum junction. These results suggest that striated muscle contains an intracellular Ca²⁺ release channel that is identical with the ryanodine receptor and the transverse-tubule-sarcoplasmic reticulum spanning feet structures.     

Inheritance behavior of information coding for small subunit polypeptides of fraction 1 protein

In various genera of plants, the small subunit of fraction 1 protein is often composed of more than one kind of polypeptide; these differ in isoelectric points and amino acid composition. Previous analysis of numerous individual progeny of Nicotiana tabacum (two kinds of polypeptides), N. glauca + N. langsdorffii parasexual hybrids (three kinds), and other examples showed no change in F-1 protein composition as a consequence of alternation of generations. Experiments reported here show that absence of one member of each of the 24 different pairs of chromosomes in an N. tabacum monosomic series and also absence of the “S” pair in a nullisome did not affect F-1 protein composition. Absence of the “E” pair caused reduction in the amount of the least acidic of the two kinds of N. tabacum small subunit polypeptides. The question of how many individual progeny of self-fertile hybrids would have to be analyzed to detect segregation of genes coding for F-1 protein small subunit polypeptides, if segregation occurs, was answered by analysis of F₁ hybrids between N. otophora and N. tomentosiformis, and two subspecies of N. suaveolens, together with their F₂ progeny. In both cases, analysis of 16 progeny was sufficient to demonstrate a segregation pattern of two F₁ hybrid type to one each of the two parental types. Therefore, in the absence of segregation, it is likely that coding information for different kinds of F-1 protein small subunit polypeptides is sequestered on heterologous chromosomes, as postulated in previous reports.     

Relating proton pumps with gap junctions: colocalization of ductin,the channel-forming subunit c of V-ATPase,with subunit a and with innexins 2 and 3 during <Emphasis Type="Italic">Drosophila</Emphasis> oogenesis

Background

Ion-transport mechanisms and gap junctions are known to cooperate in creating bioelectric phenomena, like pH gradients, voltage gradients and ion fluxes within single cells, tissues, organs, and whole organisms. Such phenomena have been shown to play regulatory roles in a variety of developmental and regenerative processes. Using Drosophila oogenesis as a model system, we aim at characterizing in detail the mechanisms underlying bioelectric phenomena in order to reveal their regulatory functions. We, therefore, investigated the stage-specific distribution patterns of V-ATPase components in relation to gap-junction proteins.

Results

We analysed the localization of the V-ATPase components ductin (subunit c) and subunit a, and the gap-junction components innexins 2 and 3, especially in polar cells, border cells, stalk cells and centripetally migrating cells. These types of follicle cells had previously been shown to exhibit characteristic patterns of membrane channels as well as membrane potential and intracellular pH. Stage-specifically, ductin and subunit a were found either colocalized or separately enriched in different regions of soma and germ-line cells. While ductin was often more prominent in plasma membranes, subunit a was more prominent in cytoplasmic and nuclear vesicles. Particularly, ductin was enriched in polar cells, stalk cells, and nurse-cell membranes, whereas subunit a was enriched in the cytoplasm of border cells, columnar follicle cells and germ-line cells. Comparably, ductin and both innexins 2 and 3 were either colocalized or separately enriched in different cellular regions. While ductin often showed a continuous membrane distribution, the distribution of both innexins was mostly punctate. Particularly, ductin was enriched in polar cells and stalk cells, whereas innexin 2 was enriched in the oolemma, and innexin 3 in centripetally migrating follicle cells. In lateral follicle-cell membranes, the three proteins were found colocalized as well as separately concentrated in presumed gap-junction plaques.

Conclusions

Our results support the notion of a large variety of gap junctions existing in the Drosophila ovary. Moreover, since ductin is the channel-forming part of a proton pump and, like the innexins, is able to form junctional as well as non-junctional membrane channels, a plethora of cellular functions could be realized by using these proteins. The distribution and activity patterns of such membrane channels are expected to contribute to developmentally important bioelectric signals.

     

Biosynthetic approach to the structure of F1-ATPase (BF1 factor) from Micrococcus lysodeikticus membranes: in vivo labeling of the polypeptides and study of their relationship

The F₁-ATPase or BF₁ factor was purified from Micrococcus lysodeikticus substrain B grown in a synthetic medium in the presence of tritiated amino acids. When analyzed in sodium dodecyl sulfate-7% polyacrylamide gels, the fresh purified preparation contained α, β, γ subunits (referred as the intrinsic subunits) and two other polypeptides (designated as X and component of relative mobility 1.0) whose status as subunits remains to be established. This overall polypeptide composition was similar to that of the F₁-ATPase isolated from the same strain grown in complex medium (J. Carreira, J. M. Andreu, M. Nieto, and E. Muñoz., 1976 Mol. Cell. Biochem.10, 67–76). The distribution of ³H-labeled amino acids into purified F₁-ATPase and its constituent polypeptides under different stages of growth was used to investigate the biosynthetic relationship between the different polypeptides. The incorporation of amino acids into purified BF₁ factor was slower than that of cytoplasmic and other membrane proteins. In isotope-dilution and chase experiments, F₁-ATPase showed one of the slowest rates of decay of the incorporated label. These results point out that F₁-ATPase of M. lysodeikticus undergoes slower turnover than the overall cytoplasmic and membrane proteins. Pulse and chase experiments allowed us to conclude that the α, β, γ subunits and the components of relative mobility 1.0 are independent with differences in their turnover and therefore do not bear any apparent relation as precursors-products. The two major subunits represent seemingly the “core” of ATPase, the β subunit behaving like the most stable component. On the other hand, the γ subunit appears to be synthesized independently from this α + β complex.     

Calcium binding to the subunit c ofE. coli ATP-synthase and possible functional implications in energy coupling

The 8-kDa subunit c of theE. coli F₀ ATP-synthase proton channel was tested for Ca⁺⁺ binding activity using a⁴⁵Ca⁺⁺ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes. The purified subunit c binds⁴⁵Ca⁺⁺ strongly with Ca⁺⁺ binding properties very similar to those of the 8-kDa CF₀ subunit III of choloroplast thylakoid membranes. The N-terminal f-Met carbonyl group seems necessary for Ca⁺⁺ binding capacity, shown by loss of Ca⁺⁺ binding following removal of the formyl group by mild acid treatment. The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca⁺⁺ binding, shown by Ca⁺⁺ binding being retained in twoE. coli mutants, Asp61Asn and Asp61Gly. The Ca⁺⁺ binding is pH dependent in both theE. coli and thylakoid 8-kDa proteins, being absent at pH 5.0 and rising to a maximum near pH 9.0. A treatment predicted to increase the Ca⁺⁺ binding affinity to its F₀ binding site (chlorpromazine photoaffinity attachment) caused an inhibition of ATP formation driven by a base-to-acid pH jump in whole cells. Inhibition was not observed when the Ca⁺⁺ chelator EGTA was present with the cells during the chlorpromazine photoaffinity treatment. An apparent Ca⁺⁺ binding constant on the site responsible for the UV plus chlorpromazine effect of near 80–100 nM was obtained using an EGTA-Ca⁺⁺ buffer system to control free Ca⁺⁺ concentration during the UV plus chlorpromazine treatment. The data are consistent with the notion that Ca⁺⁺ bound to the periplasimic side of theE. coli F₀ proton channel can block H⁺ entry into the channel. A similar effect occurs in thylakoid membranes, but the Ca⁺⁺ binding site is on the lumen side of the thylakoid, where Ca⁺⁺ binding can modulate acid-base jump ATP formation. The Ca⁺⁺ binding to the F₀ and CF₀ complexes is consistent with a pH-dependent gating mechanism for control of H⁺ ion flux across the opening of the H⁺ channel.This work was supported in part by grants from the Department of Energy and the U.S. Department of Agriculture.On leave from the Institute of Soil Science and Photosynthesis, Russian Academy of Science, Pushchino, Russia.     

Characterization of a highly diverged mitochondrial ATP synthase Fo subunit in Trypanosoma brucei

The mitochondrial F₁F_o ATP synthase of the parasite Trypanosoma brucei has been previously studied in detail. This unusual enzyme switches direction in functionality during the life cycle of the parasite, acting as an ATP synthase in the insect stages, and as an ATPase to generate mitochondrial membrane potential in the mammalian bloodstream stages. Whereas the trypanosome F₁ moiety is relatively highly conserved in structure and composition, the F_o subcomplex and the peripheral stalk have been shown to be more variable. Interestingly, a core subunit of the latter, the normally conserved subunit b, has been resistant to identification by sequence alignment or biochemical methods. Here, we identified a 17 kDa mitochondrial protein of the inner membrane, Tb927.8.3070, that is essential for normal growth, efficient oxidative phosphorylation, and membrane potential maintenance. Pull-down experiments and native PAGE analysis indicated that the protein is both associated with the F₁F_o ATP synthase and integral to its assembly. In addition, its knockdown reduced the levels of F_o subunits, but not those of F₁, and disturbed the cell cycle. Finally, analysis of structural homology using the HHpred algorithm showed that this protein has structural similarities to F_o subunit b of other species, indicating that this subunit may be a highly diverged form of the elusive subunit b.     

Phosphorylation of a peptide related to subunit c of the F0F1-ATPase/ATP synthase and relationship to permeability transition pore opening in mitochondria

A phosphorylated polypeptide (ScIRP) from the inner membrane of rat liver mitochondria with an apparent molecular mass of 3.5 kDa was found to be immunoreactive with specific antibodies against subunit c of F₀F₁-ATPase/ATP synthase (Azarashvily, T. S., Tyynelä, J., Baumann, M., Evtodienko, Yu. V., and Saris, N.-E. L. (2000). Biochem. Biophys. Res. Commun. 270, 741–744. In the present paper we show that the dephosphorylation of ScIRP was promoted by the Ca²⁺-induced mitochondrial permeability transition (MPT) and prevented by cyclosporin A. Preincubation of ScIRP isolated in its dephosphorylated form with the mitochondrial suspension decreased the membrane potential (_M) and the Ca²⁺-uptake capacity by promoting MPT. Incorporation of ScIRP into black-lipid membranes increased the membrane conductivity by inducing channel formation that was also suppressed by antibodies to subunit c. These data indicate that the phosphorylation level of ScIRP is influenced by the MPT pore state, presumably by stimulation of calcineurin phosphatase by the Ca²⁺ used to induce MPT. The possibility of ScIRP being part of the MPT pore assembly is discussed in view of its capability to induced channel activity.     

Compaction of a Prokaryotic Signal-Anchor Transmembrane Domain Begins within the Ribosome Tunnel and Is Stabilized by SRP during Targeting

Cotranslational targeting of membrane proteins is mediated by the universally conserved signal recognition particle (SRP). In eukaryotes, SRP attenuates translation during targeting; however, in prokaryotes, a simplified SRP is believed to carry out targeting during continuing translation. Here, we show a detailed stepwise analysis of the targeting of subunit c of the F₀ component of the bacterial ATP synthase (F₀c) to the inner membrane. We show that the first transmembrane (TM) signal-anchor domain of F₀c forms a compacted structure within the distal portion of the ribosome tunnel. This structure is formed just prior to the interaction with SRP. In the absence of SRP this structure is lost as the TM domain exits the tunnel; however in the presence of SRP it is stabilized. Our results suggest differences in early protein folding of substrates for prokaryotic SRP‐dependent membrane protein targeting pathways, from that of eukaryotic SRP targeting. These results imply that early TM domain recognition by targeting factors acts to ensure that the efficiency of membrane targeting is maintained.     

Ryanodine as a functional probe of the skeletal muscle sarcoplasmic reticulum Ca2+ release channel

Ryanodine is a neutral plant alkaloid which functions as a probe for an intracellular Ca²⁺ release channel (ryanodine receptor) in excitable tissues. Using [³H]ryanodine, a 30 S protein complex comprised of four polypeptides of M_r 565,000 has been isolated and functionally reconstituted into planar lipid bilayers. The effects of salt concentration and divalent cations on skeletal muscle sarcoplasmic reticulum [³H]ryanodine binding and Ca²⁺ release channel activity have been compared. These studies suggest that ryanodine is a good probe for investigating the function of the release channel.     

Analysis of myelin proteolipid protein and F0ATPase subunit 9 in normal andjimpy CNS

Membrane fractions and chloroform-methanol (C-M) extracts ofjimpy (jp) and normal CNS at 17–20 days were examined by immunoblot and sequence analysis to determine whether myelin proteolipid protein (PLP) or DM-20 could be detected in jp CNS. No reactivity was detected in jp samples with several PLP antibodies (Abs) except with one Ab to amino acids 109–128 of normal PLP. Proteins in the immunoreactive bands 26 Mr comigrating with PLP were sequenced for the first 10–12 residues. A sequence corresponding to PLP was found in normal CNS, as expected, but not in the band from jp CNS. Our results provide no evidence for an aberrant form of PLP in jp CNS at 17–20 days. This and other studies suggest that the abnormalities in jp brain are not due to toxicity of the mutant jp PLP/DM-20 proteins. Interestingly, a sequence identical to the amino terminus of the mature proton channel subunit 9 of mitochondrial F₀ ATPase was detected in the immunoreactive bands 26 Mr in both normal and jp samples. This identification was supported by reactivity with an Ab to the F₀ subunit and by labeling with dicyclohexylcarbodiimide (DCCD). In contrast to PLP isolated from whole CNS, PLP isolated from myelin was devoid of F₀ subunit 9 based on sequence analysis and lack of reactivity with an Ab to the F₀ subunit, yet still reacted with DCCD. This finding rules out the possibility that contaminating F₀ ATPase gives rise to the DCCD binding exhibited by PLP and confirms the possibility that PLP has proton channel activity, as suggested by Lin and Lees (1,2).Abbreviations used Ab antibody - CM conditioned medium - C M chloroform-methanol - DCCD dicyclohexylcarbodiimide - jp jimpy - Mr mobility (apparent m.w×10^–3) - PLP proteolipid protein - PVDF polyvinylidene difluoride