The determination of the separate Ca pump protein and phospholipid profile structures within reconstituted sarcoplasmic reticulum membranes via X-ray and neutron diffraction

We have previously compared the electron density profiles for several highly-functional reconstituted sarcoplasmic reticulum membranes with that for the isolated sarcoplasmic reticulum membrane (Herbette, L., Scarpa, A., Blasie, J.K., Wang, C.T., Saito, A. and Fleischer, S. (1981) Biophys. J. 36, 47–72). In this paper, we compare the separate calcium pump protein profile within these reconstituted sarcoplasmic reticulum membranes, as derived by X-ray and neutron diffraction methods, with that within isolated sarcoplasmic reticulum membranes. In addition, the time-average perturbation of the lipid bilayer by the incorporated calcium pump protein within these reconstituted sarcoplasmic reticulum membranes has been determined in some detail.     

The determination of the separate Ca2+ pump protein and phospholipid profile structures within reconstituted sarcoplasmic reticulum membranes via X-ray and neutron diffraction

We have previously compared the electron density profiles for several highly-functional reconstituted sarcoplasmic reticulum membranes with that for the isolated sarcoplasmic reticulum membrane (Herbette, L., Scarpa, A., Blasie, J.K., Wang, C.T., Saito, A. and Fleischer, S. (1981) Biophys. J. 36, 47–72). In this paper, we compare the separate calcium pump protein profile within these reconstituted sarcoplasmic reticulum membranes, as derived by X-ray and neutron diffraction methods, with that within isolated sarcoplasmic reticulum membranes. In addition, the time-average perturbation of the lipid bilayer by the incorporated calcium pump protein within these reconstituted sarcoplasmic reticulum membranes has been determined in some detail.     

The determination of the separate Ca2+ pump protein and phospholipid profile structures within reconstituted sarcoplasmic reticulum membranes via X-ray and neutron diffraction

We have previously compared the electron density profiles for several highly-functional reconstituted sarcoplasmic reticulum membranes with that for the isolated sarcoplasmic reticulum membrane (Herbette, L., Scarpa, A., Blasie, J.K., Wang, C.T., Saito, A. and Fleischer, S. (1981) Biophys. J. 36, 47-72). In this paper, we compare the separate calcium pump protein profile within these reconstituted sarcoplasmic reticulum membranes, as derived by X-ray and neutron diffraction methods, with that within isolated sarcoplasmic reticulum membranes. In addition, the time-average perturbation of the lipid bilayer by the incorporated calcium pump protein within these reconstituted sarcoplasmic reticulum membranes has been determined in some detail.     

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.     

The separate profile structures of the functional calcium pump protein and the phospholipid bilayer within isolated sarcoplasmic reticulum membranes determined by X-ray and neutron diffraction

The detailed profile structure of the isolated sarcoplasmic reticulum membrane was studied utilizing a combination of X-ray and neutron diffraction. The water and lipid profile structures within the sarcoplasmic reticulum membrane were determined at 28 A resolution directly by neutron diffraction and selective deuteration of the water and lipid components. The previously determined electron density profile structure of the sarcoplasmic reticulum membrane at 12 A resolution was subjected to model refinement analysis constrained by the neutron diffraction results, thereby providing unique higher resolution calculated lipid and protein profile structures. It was found that the lipid bilayer profile structure of the isolated sarcoplasmic reticulum membrane is asymmetric, primarily the result of more lipid residing in the inner versus the outer monolayer of the sarcoplasmic reticulum lipid bilayer. The asymmetry in the lipid composition was necessarily coincident with a complimentary asymmetry in the protein mass distribution between the two monolayers in order to preserve the overall cross-sectional area of lipid and protein throughout the lipid bilayer region of the sarcoplasmic reticulum membrane profile structure. Approximately 50% of the mass of the total protein was found to be localized externally to the sarcoplasmic reticulum membrane lipid bilayer protruding from the outer lipid monolayer into the extravesicular medium. The structural features of the protein protrusion appear to be rather variable depending upon the environment of the sarcoplasmic reticulum membrane. This highly asymmetric structural organization of the sarcoplasmic reticulum membrane profile is consistent with its primary function of unidirectional calcium transport.     

Short-chain lecithin/long-chain phospholipid unilamellar vesicles: asymmetry, dynamics, and enzymatic hydrolysis of the short-chain component

Asymmetric unilamellar vesicles are produced when short-chain phospholipids (fatty acyl chain lengths of 6-8 carbons) are mixed with long-chain phospholipids (fatty acyl chain lengths of 14 carbons or longer) in ratios of 1:4 short-chain/long-chain component. Short-chain lecithins are preferentially distributed on the outer monolayer, while a short-chain phosphatidylethanolamine derivative appears to localize on the inner monolayer of these spontaneously forming vesicles. Lanthanide NMR shift experiments clearly show a difference in head-group/ion interactions between the short-chain and long-chain species. Two-dimensional 1H NMR studies reveal efficient spin diffusion networks for the short-chain species embedded in the long-chain bilayer matrix. The short-chain lecithin is considerably more mobile than the long-chain component but has hindered motion compared to short-chain lecithin micelles. This differentiation in physical characteristics of the two phospholipid components is critical to understanding the activity of phospholipases toward these binary systems.     

Membrane Asymmetry in Isolated Canine Cardiac Sarcoplasmic Reticulum: Comparison with Skeletal Muscle Sarcoplasmic Reticulum

Cardiac sarcoplasmic reticulum (CSR), isolated from dog hearts, was shown to be asymmetric in the distribution of phospholipids across the CSR bilayer. Phosphatidylethanolamine was mostly resident in the outer leaflet, phosphatidylcholine was equally distributed across both monolayers and phosphatidylserine was found primarily in the inner monolayer. This distribution of headgroups is similar to that found in fast skeletal muscle sarcoplasmic reticulum (SSR); however, the asymmetry in CSR is not as striking as that in SSR. Phospholipids retained by the CSR calcium pump protein (CaATPase) after detergent ``stripping' were similar to those intimate to the SSR CaATPase, although the percentages of unsaturated phospholipids and plasmalogenic phospholipids are not as great as in the skeletal system. Lipids associated with the CSR CaATPase following DFDNB cross-linking showed a preference for retention of the aminophospholipids, again similar to the SSR CaATPase. Because the nonrandom distribution of membrane lipids modifies SSR function, it is likely these membrane lipids impact in situ the function of the CSR. Received: 19 December 1997/Revised: 3 April 1998     

Effect of lipid membrane structure on the adenosine 5'-triphosphate hydrolyzing activity of the calcium-stimulated adenosinetriphosphatase of sarcoplasmic reticulum

An active Ca2+-stimulated, Mg2+-dependent adenosinetriphosphatase (Ca2+-ATPase) isolated from rabbit skeletal muscle sarcoplasmic reticulum membranes has been incorporated into dilauroyl-, dimyristoyl-, dipentadecanoyl-, dipalmitoyl-, and palmitoyloleoylphosphatidylcholine bilayers by using a newly developed lipid-substitution procedure that replaces greater than 99% of the endogenous lipid. Freeze--fracture electron microscopy showed membranous vesicles of homogeneous size with symmetrically disposed fracture-face particles. Diphenylhexatriene fluorescence anisotropy was used to define the recombinant membrane phase behavior and revealed more than one transition in the membranes. Enzymatic analysis indicated that saturated phospholipid acyl chains inhibited both overall ATPase activity and Ca2+-dependent phosphoenzyme formation below the main lipid phase transition temperature (Tm) of the lipid-replaced membranes. At temperatures above Tm, ATPase activity but not phosphoenzyme formation was critically dependent on acyl chain length and thus bilayer thickness. No ATPase activity was observed in dilauroylphosphatidylcholine bilayers. Use of the nonionic detergent dodecyloctaoxyethylene glycol monoether demonstrated that the absence of activity was not due to irreversible inactivation of the enzyme. Increased bilayer thickness resulted in increased levels of activity. An additional 2-fold rise in activity was observed when one of the saturated fatty acids in dipalmitoylphosphatidylcholine was replaced by oleic acid, whose acyl chain has a fully extended length comparable to that of palmitic acid. These results indicate that the Ca2+-ATPase requires for optimal function a "fluid" membrane with a minimal bilayer thickness and containing unsaturated phospholipid acyl chains.     

Structure-function studies of canine cardiac sarcolemmal membranes. II. Structural organization of the sarcolemmal membrane as determined by electron microscopy and lamellar X-ray diffraction

The morphological and ultrastructural properties of highly purified canine cardiac sarcolemmal vesicles, prepared by a modification (Colvin, R.A., Ashavaid, T.F. and Herbette, L.G. (1985) Biochim. Biophys. Acta 812, 601-608) of the method of Jones et al. (Jones L.R., Madlock, S.W. and Besch, H.R. (1980) J. Biol. Chem. 255, 9971-9980), were examined by several techniques. Thin-section electron microscopy showed predominantly intact unilamellar vesicles with little staining beyond the lipid bilayer boundaries. Freeze-fracture electron microscopy demonstrated that the majority of particles are approx. 90 A diameter and present at a density of 780 +/- 190 micrometers-2 (+/- S.D.). If it is assumed that some of these particles represent the (Na+ + K+)-ATPase, the finding that they are largely confined to the convex fracture face suggests a predominant right-side-out orientation of these sarcolemmal vesicles that is consistent with biochemical assays. The sarcolemmal membrane width measured by electron microscopy (unhydrated membrane width of 50-70 A) is consistent with the unit cell dimensions of 56-77 A determined by lamellar X-ray diffraction (hydrated membrane width). A unit cell dimension of 56-62 A was also found by X-ray diffraction for sarcolemmal lipids extracted from these preparations, indicating that the isolated sarcolemmal preparations do not contain a significant surface coat (glycocalyx). As both cardiac and skeletal sarcoplasmic reticulum membranes have a 80-100 A membrane width, these findings demonstrate that the purified sarcolemmal membrane is structurally distinct from both cardiac and skeletal sarcoplasmic reticulum. In contrast to the protein-rich skeletal sarcoplasmic reticulum membrane, which contains a single essential protein responsible for the regulation of cytosolic Ca2+ concentration, the sarcolemma is a lipid-rich membrane that contains a variety of proteins associated with many regulatory functions served by this membrane in cardiac muscle.     

Asymmetric distribution of phosphatidylethanolamine fatty acyl chains in the membrane of vesicular stomatitis virus

The membrane of vesicular stomatitis virus (VSV) contains two distinct pools of phosphatidylethanolamine molecules which reside in the inner and outer phospholipid monolayers, respectively. 36% of the total membrane phosphatidylethanolamine is found in the outer monolayer while 64% is found in the inner. The two pools of VSV phosphatidylethanolamine can be distinguished operationally by the fact that only outer phosphatidylethanolamine is reactive in intact virions with the membrane-impermeable reagent trinitrobenzenesulfonate (TNBS). We have made use of this property to separate inner from outer VSV phosphatidylethanolamine and to determine the fatty acyl chain compositions of the two phosphatidylethanolamine pools separately. The results show that compared to outer phosphatidylethanolamine, inner phosphatidylethanolamine molecules contain a significantly higher proportion of unsaturated fatty acyl chains. Furthermore, whereas the proportion of unsaturated fatty acyl chains was found to be quite similar at the 1 and 2 glycerol carbon atoms in inner phosphatidylethanolamine, a marked dissimilarity was observed in outer phosphatidylethanolamine; outer phosphatidylethanolamine was enriched in saturated fatty acyl chains at the 1 position and in unsaturated fatty acyl chains at the 2 position. The differential fatty acyl chain composition of inner compared to outer phosphatidylethanolamine indicates that rapid, random transmembrane migration (flip-flop) of phosphatidylethanolamine does not occur in the VSV membrane. The nature of the fatty acyl chain asymmetry observed in VSV phosphatidylethanolamine does not support the view that the     

Asymmetric distribution of phosphatidylethanolamine fatty acyl chains in the membrane of vesicular stomatitis virus.

The membrane of vesicular stomatitis virus (VSV) contains two distinct pools of phosphatidylethanolamine molecules which reside in the inner and outer phospholipid monolayers, respectively. 36% of the total membrane phosphatidylethanolamine is found in the outer monolayer while 64% is found in the inner. The two pools of VSV phosphatidylethanolamine can be distinguished operationally by the fact that only outer phosphatidylethanolamine is reactive in intact virions with the membrane-impermeable reagent trinitrobenzenesulfonate (TNBS). We have made use of this property to separate inner from outer VSV phosphatidylethanolamine and to determine the fatty acyl chain compositions of the two phosphatidylethanolamine pools separately. The results show that compared to outer phosphatidylethanolamine, inner phosphatidylethanolamine molecules contain a significantly higher proportion of unsaturated fatty acyl chains. Furthermore, whereas the proportion of unsaturated fatty acyl chains was found to be quite similar at the 1 and 2 glycerol carbon atoms in inner phosphatidylethanolamine, a marked dissimilarity was observed in outer phosphatidylethanolamine; outer phosphatidylethanolamine was enriched in saturated fatty acyl chains at the 1 position and in unsaturated fatty acyl chains at the 2 position. The differential fatty acyl chain composition of inner compared to outer phosphatidylethanolamine indicates that rapid, random transmembrane migration (flip-flop) of phosphatidylethanolamine does not occur in the VSV membrane. The nature of the fatty acyl chain asymmetry observed in VSV phosphatidylethanolamine does not support the view that the identity of the fatty acyl chains can uniquely specify or determine which side of the membrane individual phosphatidylethanolamine molecules come to occupy. Although fatty acyl chain asymmetry and phosphatidylethanolamine asymmetry are correlated in VSV, no simple rules can be discerned which uniquely relate the two paramaters.     

31P and 2H NMR studies of structure and motion in bilayers of phosphatidylcholine and phosphatidylethanolamine

The structural and motional properties of mixed bilayers of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) have been examined by using wide-line 31P, 14N, and 2H NMR. 2H and 14N NMR data showed that in mixed bilayers containing both PC and PE the conformations of the head-group moieties are essentially identical with those observed for bilayers containing a single phospholipid species. Equimolar amounts of cholesterol induce also only a small change in head-group conformation. 31P T1 relaxation measurements (at 300 MHz) at various temperatures of bilayers containing phospholipids with a mixture of phosphocholine and phosphoethanolamine head-groups and unsaturated fatty acid residues revealed in all cases a clearly defined minimum corresponding to the condition omega O tau C-1 approximately 1. For all phospholipid mixtures studied, the 31P T1 relaxation was homogeneous over the whole powder spectrum and could be fitted to a single-exponential decay. The 31P vs temperature profiles were analyzed by a simple correlation model following the analysis of Seelig et al. (1981) [Seelig, J., Tamm, L., Hymel, L., & Fleischer, S. (1981) Biochemistry 20, 3922-3932]. Rotational diffusion of the phosphate moiety in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was slower than that of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and the activation energy was increased by a factor of 1.7 to 31.4 kJ mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photolabeling of the integral proteins of skeletal muscle sarcoplasmic reticulum: comparison of junctional and nonjunctional membrane fractions

Rabbit skeletal muscle sarcoplasmic reticulum (SR) was fractionated by isopycnic density gradient centrifugation into longitudinal tubules (LSR) and terminal cisternae (TC). Junctional face membranes (JFM) were obtained by Triton X-100 treatment of the TC fraction (Costello, B., Chadwick, C., Saito, A., Chu, A., Maurer, A. and Fleischer, S. (1986) J. Cell Biol. 103, 741-753). Photoactivatable phospholipid analogs were introduced into LSR, TC, and JFM fractions to specifically label integral membrane proteins. Remarkably different labeling patterns were observed. Proteins of the following Mr were labeled and identified in the junctional sarcoplasmic reticulum (JFM): 350,000, 325,000, 80,000, 49,000, 37,000, 32,000, 30,000, and 6000. Polypeptides of Mr 105,000 (Ca2+-dependent ATPase), 77,000, 55,000, 41,000, 22,000, and 9000 (proteolipid) were labeled and found to be selectively localized in the nonjunctional sarcoplasmic reticulum (LSR). Calsequestrin, a key protein responsible for Ca2+ storage within the SR lumen, was never labeled, whether 1 mM CaCl2 was present or absent, and is termed a nonintegral membrane protein.     

Sarcolipin, the "proteolipid" of skeletal muscle sarcoplasmic reticulum, is a unique, amphipathic, 31-residue peptide.

The sarcoplasmic reticulum of rabbit skeletal muscle contains a small "proteolipid," i.e., a protein which is soluble in acidic CHCl3/CH3OH. We propose the name sarcolipin for this small protein, to signify its lipid-like solubility and association with the sarcoplasmic reticulum. We have determined the following amino acid sequence for sarcolipin, using protein chemistry methods: M E R S T R E L C L N F T V V L I T V I L I W L L V R S Y Q Y. This 31-residue sequence includes a 19-residue hydrophobic segment which probably spans the sarcoplasmic reticulum membrane. The molecular weight calculated from the sequence, 3733, agrees with that measured by fast atom bombardment mass spectrometry, showing that sarcolipin contains no attached fatty acyl or other prosthetic groups.     

Mechanism of the apparent regulation of Escherichia coli unsaturated fatty acid synthesis by exogenous oleic acid.

Starvation of strains of Escherichia coli which are glycerol auxotrophs and are also defective in beta oxidation results in the accumulation of large amounts of free fatty acid (Cronan, J. E., Jr., Weisberg, L. W., and Allen, R. G. (1975) J. Biol. Chem. 250, 5835-5840). We now report that addition of exogenous oleic acid to these cultures results in no decrease in the synthesis of the unsaturated acids of the free fatty acid fraction although a 40 to 60% decrease of [14C]acetate incorporation into phospholipid unsaturated acyl moieties occurs under these conditions. This result indicates that the decreased synthesis of phospholipid unsaturated acyl moieties observed by others during oleic acid supplementation can be attributed to competition between exogenous and endogenously synthesized unsaturated fatty acids rather than a curtailment of unsaturated fatty acid synthesis per se.     

Spontaneous calcium release from sarcoplasmic reticulum. A re-examination.

A recent study by Blayney and co-workers (Blayney, L., Thomas, H., Muir, J. and Henderson, A. (1977) Biochim. Biophys. Acta 470, 128--133) purported to demonstrate that apparent spontaneous calcium release in sarcoplasmic reticulum is an artifact of the uptake of murexide dye. This report demonstrates that spontaneous calcium release (1) takes place despite equilibration of murexide sarcoplasmic reticulum to a stable baseline; (2) may be reversed by addition of ATP or oxalate after release has begun. The identical phenomenon can be demonstrated utilizing the indicator arsenazo III or Millipore filtration methods. The results suggest that equilibration of the murexide with sarcoplasmic reticulum vesicles must occur prior to ATP addition in order to achieve a stable baseline but that spontaneous calcium release is not an artifact.     

Effects of potassium on lipid-protein interactions in light sarcoplasmic reticulum

This study shows the effect of K+ on phospholipid-protein interactions in light sarcoplasmic reticulum (LSR) as measured by 31P NMR. In the presence of 110 mM K+, a substantial effect of the membrane protein on the behavior of the phospholipids was detected. Subtracting the spectrum of the LSR lipid extract from the spectrum of the intact LSR membrane produced a difference spectrum of much greater breadth than the normal phospholipid bilayer powder pattern. This powder pattern is indicative of a phospholipid domain considerably more motionally restricted than the phospholipids in a normal phospholipid bilayer. The apparent axially symmetric powder pattern is consistent with axial diffusion. In a reconstituted membrane containing the calcium pump protein at a lipid/protein ratio much less than in the light sarcoplasmic reticulum, the broad component was more prominent. The relative resonance intensity of the broad component appeared to be proportional to the lipid/protein ratio of the membrane. In 10 mM K+, no broad powder pattern is observed in the corresponding difference spectrum. Thus, in the absence of potassium, the membrane protein has much less influence on the phospholipid of the membrane, as measured by 31P NMR. In addition to the effects of K+ on the membrane structure of the sarcoplasmic reticulum, K+ modulated the function of the calcium pump. The rate of calcium-dependent ATP hydrolysis increased in light sarcoplasmic reticulum when [K+] increased from 10 to 110 mM. The rate of calcium transport was also stimulated by an increase in K+.     

Unsaturated aminophospholipids are preferentially retained by the fast skeletal muscle CaATPase during detergent solubilization. Evidence for a specific association between aminophospholipids and the calcium pump protein.

When fast twitch skeletal muscle vesicles (SR) and purified calcium pump protein are stripped with the nonionic detergent C12E8 (octaethylene glycol dodecyl ether), not all the membrane phospholipids are removed from the calcium pump protein. Maximal extraction produces a remnant of 6-8 mol of phospholipid/mole of calcium ATPase (CaATPase). In contrast to native SR and the prestripped purified CaATPase, the remaining phospholipid is markedly enriched in phosphatidylethanolamine (PE) and phosphatidylserine (PS) in both preparations; the remaining lipid is also enriched in phospholipid that is predominantly unsaturated. In addition, virtually all of the associated PE is plasmalogenic (96% as opposed to 63% in the native SR). The amino-specific cross-linking reagent DFDNB (1,5-difluoro-2,4-dinitrobenzene sulfonic acid) and the amino binding reagent TNBS (2,4,6-trinitrobenzene sulfonic acid) were utilized to identify the monolayer of the native preparation where these phospholipids reside, and to determine which phospholipids are closely associated with the calcium pump protein following detergent treatment. These studies demonstrate that PE and PS are closely associated with the pump protein, PE residing almost exclusively in the outer monolayer of SR, while PS resides in the inner monolayer. Nonspecific phospholipid exchange protein was shown to be capable of exchanging phospholipids from donor vesicles into those phospholipids associated with the CaATPase; stripping of lipid-exchanged vesicles with C12E8 exhibited the same specificity with regard to head-group species (i.e., PE is markedly enriched in the extracted protein associated fraction). The results suggest that specific protein-lipid interactions exist, favoring the association of plasmalogenic aminophospholipids with the calcium pump protein.     

Structural comparison of metarhodopsin II, metarhodopsin III, and opsin based on kinetic analysis of Fourier transform infrared difference spectra.

The "membrane bilayer" pathway (Rhodes, D. G., J. G. Sarmiento, and L. G. Herbette. 1985. Mol. Pharmacol. 27:612-623.) for 1,4-dihydropyridine calcium channel drug (DHP) binding to receptor sites in cardiac sarcolemmal membranes has been extended to include the interaction of amphiphiles within the lipid bilayer. These studies focused on the ability of the Class III antiarrhythmic agents bretylium and clofilium to nonspecifically inhibit DHP-receptor binding in canine cardiac sarcolemma. Clofilium was found to inhibit nimodipine binding with an inhibition constant of approximately 5 microM, whereas bretylium had no effect on nimodipine binding. Small angle x-ray diffraction was then used to examine the differential ability of these two Class III agents to inhibit DHP-receptor binding. The time-averaged locations of bretylium, clofilium, and nimodipine in bovine cardiac phosphatidylcholine (BCPC) bilayers (supplemented with 13 mol% cholesterol) were determined to a resolution of 9 A. The location of bretylium as dominated by its phenyl ring in BCPC bilayers was found to be at the hydrocarbon core/water interface, similar to that of the dihydropyridine ring of nimodipine. The location of clofilium as dominated by its phenyl ring was found to be below the hydrocarbon/core water interface within the hydrocarbon chain region of the bilayer, similar to that of the phenyl ring of nimodipine. The location of the dihydropyridine ring portion of nimodipine has previously been shown by neutron diffraction to be located at the hydrocarbon core/water interface of native sarcoplasmic reticulum, consistent with the small angle x-ray data from model membranes in this paper. Therefore, we speculate that the nonspecific inhibition arises from the interaction of clofilium's phenyl ring with the site on the calcium channel receptor where the phenyl ring portion of nimodipine must interact. The DHP-receptor binding pathway would then involve both nonspecific (membrane) and specific (protein) binding components, both of which are necessary for receptor binding.     

Functional characterization of junctional terminal cisternae from mammalian fast skeletal muscle sarcoplasmic reticulum

Junctional terminal cisternae are a recently isolated sarcoplasmic reticulum fraction containing two types of membranes, the junctional face membrane with morphologically intact "feet" structures and the calcium pump membrane [Saito, A., Seiler, S., Chu, A., & Fleischer, S. (1984) J. Cell Biol. 99, 875-885]. In this study, the Ca2+ fluxes of junctional terminal cisternae are characterized and compared with three other well-defined fractions derived from the sarcotubular system of fast-twitch skeletal muscle, including light and heavy sarcoplasmic reticulum, corresponding to longitudinal and terminal cisternae regions of the sarcoplasmic reticulum, and isolated triads. Functionally, junctional terminal cisternae have low net energized Ca2+ transport measured in the presence or absence of a Ca2+-trapping anion, as compared to light and heavy sarcoplasmic reticulum and triads. Ca2+ transport and Ca2+ pumping efficiency can be restored to values similar to those of light sarcoplasmic reticulum with ruthenium red or high [Mg2+]. In contrast to junctional terminal cisternae, heavy sarcoplasmic reticulum and triads have higher Ca2+ transport and are stimulated less by ruthenium red. Heavy sarcoplasmic reticulum appears to be derived from the nonjunctional portion of the terminal cisternae. Our studies indicate that the decreased Ca2+ transport is referable to the enhanced permeability to Ca2+, reflecting the predominant localization of Ca2+ release channels in junctional terminal cisternae. This conclusion is based on the following observations: The Ca2+, -Mg2+ -dependent ATPase activity of junctional terminal cisternae in the presence of a Ca2+ ionophore is comparable to that of light sarcoplasmic reticulum when normalized for the calcium pump protein content; i.e., the enhanced Ca2+ transport cannot be explained by a faster turnover of the pump. Ruthenium red or elevated [Mg2+] enhances energized Ca2+ transport and Ca2+ pumping efficiency in junctional terminal cisternae so that values approaching those of light sarcoplasmic reticulum are obtained. Rapid Ca2+ efflux in junctional terminal cisternae can be directly measured and is blocked by ruthenium red or high [Mg2+]. Ryanodine at pharmacologically significant concentrations blocks the ruthenium red stimulation of Ca2+ loading. Ryanodine binding in junctional terminal cisternae, which appears to titrate Ca2+ release channels, is 2 orders of magnitude lower than the concentration of the calcium pump protein. By contrast, light sarcoplasmic reticulum has a high Ca2+ loading rate and slow Ca2+ efflux that are not modulated by ruthenium red, ryanodine, or Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)