Improved experimental limit on the electric dipole moment of the neutron.

An experimental search for an electric dipole moment (EDM) of the neutron has been carried out at the Institut Laue-Langevin, Grenoble. Spurious signals from magnetic-field fluctuations were reduced to insignificance by the use of a cohabiting atomic-mercury magnetometer. Systematic uncertainties, including geometric-phase-induced false EDMs, have been carefully studied. The results may be interpreted as an upper limit on the neutron EDM of |d(n)|< 2.9 x 10(-26)e cm (90% C.L.).

Dystrophin associates with caveolae of rat cardiac myocytes: relationship to dystroglycan.

The possibility of an interaction between the cytoskeletal protein dystrophin and cell surface caveolae in the mammalian myocardium was investigated by several techniques. Caveolin (cav)-3-enriched, detergent-insoluble membranes isolated from purified ventricular sarcolemma by density-gradient fractionation were found to contain dystrophin and dystroglycan. Further purification of cav-3-containing membranes by immunoprecipitation using anti-cav-3-coated magnetic beads yielded dystrophin but not always dystroglycan. Electron microscopic analysis of precipitated material revealed caveola-sized vesicular profiles that could be double-labeled with anti-dystrophin and anti-cav-3 antibodies. In contrast, immunoprecipitation of membranes with anti-dystrophin-coated beads yielded both cav-3 and dystroglycan. Electron microscopic analysis of this material showed heterogeneous membrane profiles, some of which could be decorated with anti-cav-3 antibodies. To confirm that dystrophin and cav-3 were closely associated in cardiac myocytes, we verified that dystrophin was also present in immunoprecipitated cav-3-containing membranes from detergent extracts, as well as in sonicated extracts of purified ventricular myocytes. Confocal immunofluorescence microscopy of ventricular and atrial cardiac myocytes showed that the cellular distributions of cav-3 and dystrophin partially overlapped. Immuno-electron micrographs of thin sections of rat atrial myocytes revealed a fraction of dystrophin molecules that are in apparently close apposition to caveolae. These results suggest that a subpopulation of dystrophin molecules interacts with cardiac myocyte caveolae in vivo and that some of the dystrophin is engaged in linking cav-3 with the dystroglycan complex.

T-cadherin is a major glycophosphoinositol-anchored protein associated with noncaveolar detergent-insoluble domains of the cardiac sarcolemma.

Sucrose-density flotation analysis of Triton-insoluble membrane domains isolated from highly purified sheep ventricular sarcolemma revealed the presence of two major 120- and 100-kDa proteins. Both species migrated in two-dimensional isoelectric focussing/SDS gels with an apparent pI of approximately 4.3, suggesting that they might be related. Microsequence analysis of peptides derived from the 100-kDa protein yielded amino acid sequences with high homology to T-cadherin, a truncated cadherin lacking a cytoplasmic domain. The similarity was confirmed using antibodies to chicken T-cadherin that reacted with both proteins on immunoblots. T-cadherin was released from the detergent-insoluble sarcolemmal fraction by phospholipase C treatment indicating that it is linked to the membrane by a glycophosphoinositol anchor. T-cadherin could be ADP-ribosylated by a transferase that was also present in the caveolin-enriched Triton-insoluble fraction. T-cadherin-containing membrane fragments cofractionated on sucrose gradients with caveolin-3, a marker protein for myocyte caveolae. However, immunopurified caveolin-3-containing membranes contained no associated T-cadherin. Immunocytochemical analysis of cultured rat atrial myocytes revealed that T-cadherin and caveolin have related but nonoverlapping staining patterns. These results suggest that T-cadherin is a major glycophosphoinositol-linked protein in cardiac myocytes and that it may be located in plasma membrane "rafts" distinct from but possibly adjacent to caveolae.

Type B atrial natriuretic peptide receptor in cardiac myocyte caveolae.

We have previously shown that atrial natriuretic peptide (ANP) is present in caveolae of in situ rat atrial myocytes. To investigate whether intracaveolar ANP of rat atrial myocytes exists within caveolae bound to type B ANP receptors (ANP-RB, a guanylyl cyclase), we have used confocal immunofluorescence microscopy applied to primary cultures of atrial myocytes from adult rats and to freshly dissociated rat atrial myocytes (not cultured). These experimental designs tested whether atrial myocyte ANP-RB colocalizes at the plasmalemma and elsewhere in the cell with the muscle-specific isoform of the caveolar coating protein caveolin-3, and with a fraction of cellular ANP. The experiments showed that cellular caveolin-3, a fraction of cellular ANP-RB, and a fraction of cellular ANP colocalize at the plasmalemma of cultured atrial myocytes and of freshly dissociated atrial myocytes. The observations support the hypothesis that in rat atrial myocytes, intracaveolar ANP is bound to ANP-RB, a protein whose cytosolic amino acid sequences are known to encode guanylyl cyclase activity. We suggest that among the (probably multiple) effects of the cGMP thus generated in the cytoplasmic microdomain underlying atrial myocyte caveolae may be the activation of cGMP-dependent protein kinase, which would thereby inhibit plasma membrane Ca2+ channel activity and contribute to a negative inotropic effect of ANP.

Quantification of ANP mRNA in primary cultures of adult rat atrial myocytes by image processing: in situ hybridization to multiple parallel samples using single-stranded cDNA probes.

In primary cultures of adult rat atrial myocytes, we quantified the accumulation of atrial natriuretic peptide (ANP) mRNA in parallel with ANP secretion. ANP mRNA was quantified by image analysis of myocytes hybridized in situ with single-stranded cDNA probes generated by two successive thermal cycling procedures. In situ analysis permitted measurement of many small experimental samples in tandem while avoiding the possibility of differential extraction and processing of mRNA from sample to sample. The single-step application of 32P-labeled probes allowed processing of many parallel samples and generated intense punctate autoradiographic signals that were readily countable by image processing. Biotin-labeled probes, in conjunction with gold-labeled anti-biotin antibodies and silver intensification, gave an apparently equivalent specific signal but presented more difficulty in uniform processing of many samples and was harder to quantify by our image processing system. Measurement of ANP mRNA during atrial myocyte culture showed that ANP mRNA accumulated from undetectable levels after 1 day of culture to maximal levels by Day 8. In contrast, secretion of ANP (which is stored in atrial granules) slowly decreased, but was not abolished, during the first 5 days of culture. Subsequently, ANP secretion increased, with the increase trailing ANP mRNA accumulation by at least 24 hr.

Fast lidocaine block of cardiac and skeletal muscle sodium channels: one site with two routes of access.

We have studied the block by lidocaine and its quaternary derivative, QX-314, of single, batrachotoxin (BTX)-activated cardiac and skeletal muscle sodium channels incorporated into planar lipid bilayers. Lidocaine and QX-314, applied to the intracellular side, appear to induce incompletely resolved, rapid transitions between the open and the blocked state of BTX-activated sodium channels from both heart and skeletal muscle. We used amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157-186.) to estimate the rate constants for block and unblock. Block by lidocaine and QX-314 from the cytoplasmic side exhibits rate constants with similar voltage dependence. The blocking rate increases with depolarization, and the unblocking rate increases with hyperpolarization. Fast lidocaine block was virtually identical for sodium channels from skeletal (rat, sheep) and cardiac (beef, sheep) muscle. Lidocaine block from the extracellular side occurred at similar concentrations. However, for externally applied lidocaine, the blocking rate was voltage-independent, and was proportional to concentration of the uncharged, rather than the charged, form of the drug. In contrast, unblocking rates for internally and externally applied lidocaine were identical in magnitude and voltage dependence. Our kinetic data suggest that lidocaine, coming from the acqueous phase on the cytoplasmic side in the charged form, associates and dissociates freely with the fast block effector site, whereas external lidocaine, in the uncharged form, approaches the same site via a direct, hydrophobic path.

State-dependent block underlies the tissue specificity of lidocaine action on batrachotoxin-activated cardiac sodium channels.

We have identified two kinetically distinct modes of block, by lidocaine, of cardiac sodium channels, activated by batrachotoxin and incorporated into planar lipid bilayers. Here, we analyze the slow blocking mode which appears as a series of nonconducting events that increase in frequency and duration with increasing lidocaine concentrations. This type of block occurred rarely, if at all, for the skeletal muscle sodium channel subtype. Kinetic analysis showed that a linear open-closed-blocked model is sufficient to account for the major features of our data. Slow block occurs from a long closed state that is a distinguishing characteristic of cardiac channels under these conditions. Slow block showed no significant voltage dependence in the range of -60 to -20 mV for which the detailed kinetic analysis was performed, and was not elicited by application of the permanently charged lidocaine derivative QX-314. By contrast, the fast block, described in the companion paper, results from drug binding to the open state, and is similar for cardiac and skeletal muscle sodium channels. Application of trypsin to the cytoplasmic end of the channel eliminates both the spontaneous, long, gating closures and slow block. Thus, the lidocaine-sensitive closed state of batrachotoxin-activated cardiac sodium channels exhibits a protease susceptibility resembling that of the inactivated state of unmodified sodium channels. It is the slow block caused by lidocaine binding to this closed state that underlies the channel-subtype specificity of lidocaine action in our experiments.

Expression of phospholamban mRNA during early avian muscle morphogenesis is distinct from that of alpha-actin.

We have studied the expression of phospholamban during the early development of chick embryos by in situ hybridization and have compared it to that of alpha-cardiac and alpha-skeletal actin. In adult cross-striated muscles there is only one phospholamban gene and it is expressed exclusively in the heart and slow muscles. In the heart phospholamban transcripts were first detected at stage 14 in the region of presumptive ventricle and at stage 20 in the atrium. In the myotomal portion of the somites phospholamban mRNA was first detected at stage 20, which lagged behind the appearance of the alpha-actins. In the limb rudiments all three mRNAs were barely detectable through stage 24, but increased by stage 28+. However, quantitative analysis of signal intensity at stage 28+ indicated that less phospholamban mRNA is present in the limb bud than in the myotome since for phospholamban the ratio of the signal density in the myotome to that in the limb rudiments was about twice the value of the ratio determined for the alpha-actins. Northern blot analysis of embryonic day 11 chick fast pectoralis muscle showed that phospholamban mRNA was not detected in vivo while alpha-cardiac actin mRNA was. Moreover, no phospholamban mRNA was detected in primary cultures derived from pectoralis muscle of the same age. In concert with previous observations that phospholamban is not detectable at stage 30-32 in wing or thigh muscle, these results suggest that phospholamban mRNA is expressed independently of the alpha-actins in the limb buds during early myogenesis.

Divalent cation competition with [3H]saxitoxin binding to tetrodotoxin-resistant and -sensitive sodium channels. A two-site structural model of ion/toxin interaction.

Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cd2+, and Zn2+) the sequence for [3H]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations, Cd2+ and Zn2+ did so at much lower concentrations. In addition, and by way of explication, the divalent ion competition curves for both brain and cardiac channels (except for Cd2+ and Zn2+ in heart and Zn2+ in brain) had slopes of less than -1, consistent with more than one interaction site. Two-site curves had statistically better fits than one-site curves. The derived values of KI for the higher affinity sites were similar between the channel types, but the lower affinity KI's were larger for heart. On the other hand, the slopes of competition curves for Cd2+ and Zn2+ were close to -1, as if the cardiac Na channel had one dominant site of interaction or more than one site with similar values for KI. pH titration of [3H]STX binding to cardiac channels showed a pKa of 5.5 and a slope of 0.6-0.9, compared with a pKa of 5.1 and slope of 1 for brain channels. Tetramethyloxonium (TMO) treatment abolished [3H]STX binding to cardiac and brain channels and STX protected channels, but the TMO effect was less dramatic for cardiac channels. Trinitrobenzene sulfonate preferentially abolished [3H]STX binding to brain channels by action at an STX protected site.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation of membranes enriched in "tetrodotoxin-insensitive" saxitoxin-binding sites from mammalian ventricle. Receptor solubilization.

Purification and characterization of Na+ channel protein from mammalian ventricular myocytes has heretofore been complicated by the low concentration of Na+ channels and by the finding that mammalian ventricles contain both tetrodotoxin (TTX)-sensitive channels (TSC), with high affinity for saxitoxin (STX), and TTX-insensitive channels (TIC), with low affinity for STX. Most (perhaps all) of the sodium current for myocardial cell action potentials is carried by TIC. Most, if not all, of the cardiac TSC reside in nerves innervating the heart. To isolate TIC in sufficient quantity for further study, we prepared t-tubular sarcolemmal vesicles from large (sheep) hearts with techniques designated to minimize contamination from nerve plasmalemma. Discontinuous sucrose density gradient centrifugation of these membranes produced membrane vesicles, some of which contained no detectable TSC (range 94-100% TIC, or 0-6% TSC), at a concentration of 200-1500 fmol total sites/mg protein, with yields of 4.0-25.0 mg protein/100 g starting material (ventricle). TTX-insensitive STX-binding sites were solubilized from the membranes by 1% digitonin (and with less stability by Triton X-100). The equilibrium binding constant and dissociation rate coefficient for STX binding to the digitonin-solubilized sites were similar to those of the binding sites for the unsolubilized membranes. Unlabeled TTX competed with [3H]STX for the site with 14 times less affinity than did unlabeled STX. Digitonin-solubilized sites had a half-life for STX binding of about 24 h. Binding could be further stabilized by addition of Mg2+ or Ca2+ and exogenous phospholipid.

Separation of cardiac plasmalemma into cell surface and T-tubular components. Distribution of saxitoxin- and nitrendipine-binding sites.

To compare surface sarcolemmal with T-tubular distributions of [3H]saxitoxin (STX)- and [3H]nitrendipine (NTD)-binding sites, we centrifuged membrane vesicles from sheep and bovine ventricles on a 10-40% linear sucrose gradient from which fractions were assayed for STX and NTD binding; for markers of surface sarcolemma (ouabain-sensitive Na,K-ATPase activity, [3H]quinuclidinyl benzilate binding); and for markers of junctional sarcoplasmic reticulum known to be preferentially associated with T-tubules (ryanodine-sensitive Ca2+ uptake, calsequestrin, an Mr 300,000 putative phosphorylatable "foot" protein, and electron microscopically visible junctional sarcoplasmic reticulum-plasmalemma complexes). We identified three distinct peaks in the sucrose gradient, each characterized by significant high and low affinity STX- and high affinity NTD-binding: Peak I (approximately 19% sucrose), highly enriched in surface sarcolemma; Peak III (approximately 36% sucrose), enriched in junctional sarcoplasmic reticulum markers and hence in junctional sarcoplasmic reticulum complexes with T-tubule; and Peak II (approximately 27% sucrose), showing greatest specific STX binding and only moderate NTD binding, enriched in T-tubular membrane, unassociated with junctional sarcoplasmic reticulum. For ventricular myocytes, the ratio NTD sites/STX sites was 2.5 for surface sarcolemma, but only approximately 1.0 for T-tubules. Unlike data published for mammalian skeletal muscle, sheep and beef cardiac NTD receptors were not significantly more concentrated in T-tubular than in surface plasmalemma.

Saxitoxin binding and "fast" sodium channel inhibition in sheep heart plasma membrane.

We compared specific [3H]saxitoxin (STX) binding to isolated sheep ventricular sarcolemmal vesicles with inhibition of maximal action potential upstroke velocity (V max) by STX and tetrodotoxin (TTX) in sheep trabeculae carneae. In sarcolemmal vesicles purified 30 to 40 times over cardiac homogenate, STX binding at 0 degrees C in Na-free solution exhibited both high-affinity sites (KD = 0.22 +/- 0.05 nM, n = 85 +/- 13 fmol/mg protein) and low-affinity sites (KD = 11 +/- 4 nM, n = 360 +/- 42). The STX-inhibition constant for V max in Tyrode solution at 37 degrees C was 280 nM. TTX was approximately 10% as effective as STX in displacing bound [3H]STX and inhibiting V max. Allowing for different experimental conditions during [3H]STX binding and V max measurements, we suggest that the low-affinity sites are physiologically relevant "fast" Na+ channels of myocardial cells. Combining morphometric data for plasmalemmal area of mammalian cardiac myocytes with n for low-affinity sites, we estimate 3.6-7.6 fast Na+ channels/micron2 plasmalemma.

Sodium channels in vertebrate hearts. Three types of saxitoxin binding sites in heart.

The affinity of saxitoxin binding to cardiac sarcolemmal and cytosolic fractions was examined across species. In amphibia (frog) the plasma membrane site demonstrated a high affinity (Kd approx. 5 X 10(-9) M) but the majority of the total sites in the homogenate appeared to be high affinity soluble sites (Kd approx. 2 X 10(-9) M). Chicken and turtle cardiac plasma membrane fractions bound [3H]saxitoxin with 500-fold less affinity (Kd values of approx. 2 X 10(-6) M). No binding was seen in the cytosol. The affinity of cardiac sarcolemmal binding in amphibians correlates quantitatively with the K0.5 for the inhibition of sodium currents. Physiological correlation of the low affinity saxitoxin sites in chicken and turtle with toxin concentrations necessary to inhibit the sodium current remains unclear. The hypothesis that frog cytosolic saxitoxin binding sites originated from sarcolemma during homogenization is examined. The presence of three types of saxitoxin binding sites in cardiac preparations supports the existence of sodium channel subtypes.

Natural abundance 13C NMR spectra of human muscle, normal and diseased.

13C NMR spectra of human surgical muscle samples have been obtained at 50 and 118 MHz. Numerous sharp peaks in the 13C spectrum have been assigned to carbon atoms of soluble metabolites and fatty acyl chains of neutral fats and membrane-bound phospholipids. Comparisons have been made of 13C NMR spectra of normal and diseased muscles after removal of neutral fat by extraction with isopentane. Creatine, lactic acid, and phospholipids are not removed from muscles by isopentane. The most striking difference between 13C NMR spectra of isopentane-extracted normal and diseased muscle samples was the size of the residual 30.5 ppm methylene carbon resonance, that is, small in normal muscles and in nonspecific muscle diseases, but large in myogenic and neurogenic muscle diseases. In addition, differences were also found between normal and diseased muscles in their creatine content and their ability to produce lactic acid. By deoxycholate treatment of isopentane-extracted diseased muscle it is estimated that about one-fifth of the total phospholipids are highly mobile. T1 and NOE measurements indicated that the differences in peak height for the 30.5 ppm resonance between normal and diseased muscle are due only to difference in the amount of highly mobile fatty acyl chains in the muscle and not due to differences in relaxation parameters. 13C NMR of isopentane-extracted muscle appears to permit differentiation of normal from diseased muscle and, within diseased muscle, grading of the severity of the disease.

Changes in the natural abundance 13C NMR spectra of intact frog muscle upon storage and caffeine contracture.

The carbons of phospholipids have limited mobility in fresh, resting, gastrocnemius frog muscle, but a population of phospholipids gains considerable mobility upon storage in the muscle or in contracture induced by caffeine. In parallel with the appearance of sharp phospholipid resonances, lactic acid also appears in the 13C NMR spectra. There is a correlation between the mobility of phospholipids and the depletion of phosphocreatine and ATP in muscle.

Saxitoxin binding sites in frog-myocardial cytosol.

Cytosolic fractions of frog heart homogenates contain large amounts of a soluble, large molecular weight protein that binds the specific neurotoxin saxitoxin with the same high affinity as does the plasma membrane. Another neurotoxin, tetrodotoxin, which ordinarily is competitive with saxitoxin, does not displace saxitoxin from the cytosolic sites or from plasma membrane-enriched vesicular fractions even when its concentration exceeds that of saxitoxin by a factor of 1000. Thus, cytosolic sites are similar to membrane sites in this respect. The vesicular fraction accounts quantitatively for the amount of saxitoxin bound by whole ventricles, so that no appreciable losses seem to occur. Therefore, the cytosolic site probably is a membrane site precursor, although other possibilities cannot be ruled out. In any case, the occurrence of a soluble molecule closely related to the sodium channel provides opportunities for further study of the structure of the sodium channel.