The large glycoprotein subunit of the skeletal muscle voltage-sensitive calcium channel. Deglycosylation and development

Deglycosylation was used to assess the size of the core polypeptide of the large alpha 2-glycoprotein subunit of the 1,4-dihydropyridine-sensitive calcium channel from rabbit skeletal muscle. The extent of glycosylation was assessed by measuring the shift in apparent molecular mass of the alpha 2 component following electrophoresis in sodium dodecyl sulphate/polyacrylamide gels, using anti-(alpha 2-subunit) monoclonal antibody staining of immunoblots. Chemical deglycosylation with trifluoromethanesulphonic acid produced a shift in apparent molecular mass of the alpha 2 component from Mr 140,000 to Mr 105,000, consistent with a carbohydrate content of approximately 25%. Enzymatic treatments were insufficient to deglycosylate the alpha 2 subunit fully, possibly due to the inaccessibility of glycosidic bonds to enzyme attack. Enzymatic deglycosylation procedures did, however, reduce the 1,4-dihydropyridine-binding activity of transverse-tubule membranes. Neuraminidase alone or together with endo-beta-N-acetylglucosaminidase (endoglycosidase F) reduced the number of sites for (+)[3H]PN 200-110 by 73 +/- 2% and 77 +/- 5% respectively, with no change in apparent dissociation constant, implying a possible role for the glycosylated subunits in the binding of 1,4-dihydropyridines to the calcium-channel complex. The development of the alpha 2 component in rat skeletal muscle was shown to be indistinguishable from the appearance of 1,4-dihydropyridine binding activity consistent with the involvement of the alpha 2 subunit in the calcium-channel complex at all stages of development.     

Dihydropyridine binding of the calcium channel complex from skeletal muscle is modulated by subunit interaction.

The dihydropyridine-binding subunit alpha 1 of the calcium channel complex from rabbit skeletal muscle can be partially depleted from the alpha 2 delta beta-complex using wheat germ agglutinin-affinity chromatography. This depletion of the alpha 1 from the other subunits leads to a loss of dihydropyridine-binding, which can be fully reconstituted by repletion of the alpha 1 with the other subunits. Reassembly of these subunits results in an increase in the Kd and Bmax of the dihydropyridine-binding indicating that the non-dihydropyridine-binding subunits influence dihydropyridine-binding. The affinity of the alpha 1 subunit for the other subunits was determined to be approximately 35 nM. Since the free alpha 1 subunit will not bind to the beta subunit alone, there is evidence, given the selective partitioning of the beta subunit to the lectin-bound subunit pool, that either beta binds with higher affinity to the alpha 2 delta-complex than to the free alpha 1 subunit or that the bound alpha 1 creates or modulates beta-binding. This indicates a functional high affinity interaction between the dihydropyridine-binding alpha 1 subunit and the alpha 2 delta beta-complex.     

The calcium channel antagonists receptor from rabbit skeletal muscle. Reconstitution after purification and subunit characterization

The Ca2+ channel antagonists receptor from rabbit skeletal muscle was purified to homogeneity. Following reconstitution into phosphatidylcholine vesicles, binding experiments with (+)[3H]PN 200-110, (-)[3H]D888 and d-cis-[3H]diltiazem demonstrated that receptor sites for the three most common Ca2+ channel markers copurified with binding stoichiometries close to 1:1:1. Sodium dodecyl sulfate gel analysis of the purified receptor showed that it is composed of only one protein of Mr 170,000 under non-reducing conditions and of two polypeptides of Mr 140,000 and 32,000 under disulfide-reducing conditions. Iodination of the protein of Mr 170,000 and immunoblots experiments with antisera directed against the different components demonstrated that the Ca2+ channel antagonists receptor is a complex of Mr 170,000 composed of a polypeptide chain of Mr 140,000 associated to one polypeptide chain of Mr 32,000 by disulfide bridges. One of the problems concerning this subunit structure of the putative Ca2+ channel was the presence of smaller polypeptide chains of Mr 29,000 and 25,000. Peptide mapping of these polypeptide chains and analysis of their cross-reactivity with sera directed against the proteins of Mr 170,000 and 32,000 demonstrated that they were degradative products of the Mr 32,000 component. Both the large (140 kDa) and the small (32 kDa) component of the putative Ca2+ channel are heavily glycosylated. At least 20-22% of their mass were removed by enzymatic deglycosylation. Finally the possibility that both the 140-kDa and 32-kDa components originate from a single polypeptide chain of Mr 170,000 which is cleaved by proteolysis upon purification is discussed.     

Single calcium channel behavior in native skeletal muscle

The purpose of this study was to use whole-cell and cell-attached patches of cultured skeletal muscle myotubes to study the macroscopic and unitary behavior of voltage-dependent calcium channels under similar conditions. With 110 mM BaCl2 as the charge carrier, two types of calcium channels with markedly different single-channel and macroscopic properties were found. One class was DHP-insensitive, had a single-channel conductance of approximately 9 pS, yielded ensembles that displayed an activation threshold near -40 mV, and activated and inactivated rapidly in a voltage-dependent manner (T current). The second class could only be well resolved in the presence of the DHP agonist Bay K 8644 (5 microM) and had a single-channel conductance of approximately 14 pS (L current). The 14-pS channel produced ensembles exhibiting a threshold of approximately -10 mV that activated slowly (tau act approximately 20 ms) and displayed little inactivation. Moreover, the DHP antagonist, (+)-PN 200-110 (10 microM), greatly increased the percentage of null sweeps seen with the 14-pS channel. The open probability versus voltage relationship of the 14-pS channel was fitted by a Boltzmann distribution with a VP0.5 = 6.2 mV and kp = 5.3 mV. L current recorded from whole-cell experiments in the presence of 110 mM BaCl2 + 5 microM Bay K 8644 displayed similar time- and voltage-dependent properties as ensembles of the 14-pS channel. Thus, these data are the first comparison under similar conditions of the single-channel and macroscopic properties of T current and L current in native skeletal muscle, and identify the 9- and 14-pS channels as the single-channel correlates of T current and L current, respectively.     

Low single channel conductance of the major skeletal muscle chloride channel, ClC-1.

We expressed the skeletal muscle chloride channel, ClC-1, in HEK293 cells and investigated it with the patch-clamp technique. Macroscopic properties are similar to those obtained after expression in Xenopus oocytes, except that faster gating kinetics are observed in mammalian cells. Nonstationary noise analysis revealed that both rat and human ClC-1 have a low single channel conductance of about 1 pS. This finding may explain the lack of single-channel data for chloride channels from skeletal muscle despite its high macroscopic chloride conductance.     

A vertebrate slow skeletal muscle actin isoform

Salmonids utilize a unique, class II isoactin in slow skeletal muscle. This actin contains 12 replacements when compared with those from salmonid fast skeletal muscle, salmonid cardiac muscle and rabbit skeletal muscle. Substitutions are confined to subdomains 1 and 3, and most occur after residue 100. Depending on the pairing, the 'fast', 'cardiac' and rabbit actins share four, or fewer, substitutions. The two salmonid skeletal actins differ nonconservatively at six positions, residues 103, 155, 278, 281, 310 and 360, the latter involving a change in charge. The heterogeneity has altered the biochemical properties of the molecule. Slow skeletal muscle actin can be distinguished on the basis of mass, hydroxylamine cleavage and electrophoretic mobility at alkaline pH in the presence of 8 m urea. Further, compared with its counterpart in fast muscle, slow muscle actin displays lower activation of myosin in the presence of regulatory proteins, and weakened affinity for nucleotide. It is also less resistant to urea- and heat-induced denaturation. The midpoints of the change in far-UV ellipticity of G-actin versus temperature are approximately 45 degrees C ('slow' actin) and approximately 56 degrees C ('fast' actin). Similar melting temperatures are observed when thermal unfolding is monitored in the aromatic region, and is suggestive of differential stability within subdomain 1. The changes in nucleotide affinity and stability correlate with substitutions at the nucleotide binding cleft (residue 155), and in the C-terminal region, two parts of actin which are allosterically coupled. Actin is concluded to be a source of skeletal muscle plasticity.     

Ryanodine stabilizes multiple conformational states of the skeletal muscle calcium release channel.

Nanomolar to micromolar ryanodine alters the gating kinetics of the Ca2+ release channel from skeletal sarcoplasmic reticulum (SR) fused with bilayer lipid membranes (BLM). In the presence of asymmetric CsCl and 100 microM CaCl2 cis, ryanodine (RY) (5-40 nM) activates the channel, increasing the open probability (po; maximum 300% of control) without changing unitary conductance (468 picosiemens (pS)). Statistical analyses of gating kinetics reveal that open and closed dwell times exhibit biexponential distributions and are significantly modified by nanomolar RY. Altered channel gating kinetics with low nanomolar RY is fully reversible and correlates well with binding kinetics of nanomolar [3H]RY with its high affinity site (Kd1 = 0.7 nM) under identical experimental conditions. RY (20-50 nM) induces occasional 1/2 conductance fluctuations which correlate with [3H]RY binding to a second site having lower affinity (Kd2 = 23 nM). RY (5-50 nM) in the presence of 500 mM CsCl significantly enhances Ca(2+)-induced Ca2+ release from actively loaded SR vesicles. Ryanodine > or = 50 nM stabilizes the channel in a 234-pS subconductance which is not readily reversible. RY (> or = 70 microM) produces a unidirectional transition from the 1/2 to a 1/4 conductance fluctuation, whereas RY > or = 200 microM causes complete closure of the channel. The RY required for stabilizing 1/4 conductance transitions and channel closure do not quantitatively correlate with [3H]RY equilibrium binding constants and is attributed to significant reduction in association kinetics with > 200 nM [3H]RY in the presence of 500 mM CsCl. These results demonstrate that RY stabilizes four discrete states of the SR release channel and supports the existence of multiple interacting RY effector sites on the channel protein.     

Caveolin-3 is a direct molecular partner of the Cav1.1 subunit of the skeletal muscle L-type calcium channel

Caveolin-3 is the striated muscle specific isoform of the scaffolding protein family of caveolins and has been shown to interact with a variety of proteins, including ion channels. Mutations in the human CAV3 gene have been associated with several muscle disorders called caveolinopathies and among these, the P104L mutation (Cav-3(P104L)) leads to limb girdle muscular dystrophy of type 1C characterized by the loss of sarcolemmal caveolin. There is still no clear-cut explanation as to specifically how caveolin-3 mutations lead to skeletal muscle wasting. Previous results argued in favor of a role for caveolin-3 in dihydropyridine receptor (DHPR) functional regulation and/or T-tubular membrane localization. It appeared worth closely examining such a functional link and investigating if it could result from the direct physical interaction of the two proteins. Transient expression of Cav-3(P104L) or caveolin-3 specific siRNAs in C2C12 myotubes both led to a significant decrease of the L-type Ca(2+) channel maximal conductance. Immunolabeling analysis of adult skeletal muscle fibers revealed the colocalization of a pool of caveolin-3 with the DHPR within the T-tubular membrane. Caveolin-3 was also shown to be present in DHPR-containing triadic membrane preparations from which both proteins co-immunoprecipitated. Using GST-fusion proteins, the I-II loop of Ca(v)1.1 was identified as the domain interacting with caveolin-3, with an apparent affinity of 60nM. The present study thus revealed a direct molecular interaction between caveolin-3 and the DHPR which is likely to underlie their functional link and whose loss might therefore be involved in pathophysiological mechanisms associated to muscle caveolinopathies.     

Identification of two distinct proteins that are immunologically related to the alpha 1 subunit of the skeletal muscle dihydropyridine-sensitive calcium channel.

The alpha 1 subunit of the dihydropyridine-sensitive calcium channel is a protein which is critical for excitation-contraction coupling and L-type calcium current in skeletal muscle. Using antibodies generated against peptides from three regions of the deduced amino acid sequence of the alpha 1 subunit, we have identified two distinct proteins in rabbit skeletal muscle. Both proteins appeared to be recognized by antibodies against the amino (N) terminus of the alpha 1 subunit sequence. One protein was also recognized by antibodies against an internal (I) region of the predicted sequence but not by antibodies against the carboxyl (C) terminus. In contrast, the other protein was recognized by antibodies against the carboxyl terminus but not by the antibodies against the internal region. We have designated these proteins pNI and pNC based on their patterns of antibody recognition. No protein was detected which was recognized by all three antibodies. pNI is the protein commonly identified as the alpha 1 subunit of the dihydropyridine-sensitive calcium channel. Of note is that pNI, which apparently lacks sequences from the predicted carboxyl tail, is the protein present in preparations which we have previously demonstrated contain dihydropyridine-sensitive calcium channel activity. pNC is herein identified as a skeletal muscle protein that is immunologically related to the alpha 1 subunit of the dihydropyridine-sensitive calcium channel. Its function is unknown. In addition to their distinct patterns of antibody recognition, pNI and pNC were also distinguishable by several other properties. pNC migrated as a protein of approximately 160 kDa in 5% sodium dodecyl sulfate-polyacrylamide gels versus approximately 165 kDa for pNI. pNI was enriched in transverse tubule membranes, whereas pNC was found to be enriched in triad and junctional sarcoplasmic reticulum membrane fractions and was not found in transverse tubule membranes. Under conditions in which pNI bound to wheat germ agglutinin-Sepharose, pNC did not bind. The results demonstrate that there are two proteins in skeletal muscle which are immunologically related to the alpha 1 subunit of the dihydropyridine-sensitive calcium channel but which are distinguishable by several biochemical and immunological characteristics.     

An insulin-stimulated cation channel in skeletal muscle. Inhibition by calcium causes oscillation.

A cation channel has been identified in the plasma membrane of skeletal muscle that oscillates open and closed in a regular manner. In an experimental system of patch-clamped reconstituted plasma membrane in phospholipid bilayers, the oscillations are calcium-dependent and constitute regular closing events due to inhibition of the channel by calcium with a Ki of 2.2 +/- 1 x 10(-6) M, followed by reopening. There are 3.7 +/- 1 calcium binding sites/channel. With sodium as the current vehicle, conductance is increased by voltage, insulin (Km = 5 +/- 0.6 x 10(-9) M), and hydrolyzable guanine nucleotides. Cyclic GMP alone with increase the conductance with a Km of 3.7 +/- 0.6 x 10(-7) M. In the absence of calcium, the unitary conductance with insulin + GTP or cGMP at 150 mM NaCl is 153 picosiemens. Sodium current is insensitive to 10(-5) M tetrodotoxin but inhibited by mu-conotoxin (Ki = 5 x 10(-8) M). These findings in the reconstituted system were verified in patch-clamped whole muscle cells where an insulin and cGMP-dependent sodium current inhibited by mu-conotoxin could be demonstrated. In the whole cell experiments, slow calcium-dependent oscillations of the sodium current were also detected.     

Calsequestrin and the calcium release channel of skeletal and cardiac muscle

Calsequestrin is by far the most abundant Ca(2+)-binding protein in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. It allows the Ca2+ required for contraction to be stored at total concentrations of up to 20mM, while the free Ca2+ concentration remains at approximately 1mM. This storage capacity confers upon muscle the ability to contract frequently with minimal run-down in tension. Calsequestrin is highly acidic, containing up to 50 Ca(2+)-binding sites, which are formed simply by clustering of two or more acidic residues. The Kd for Ca2+ binding is between 1 and 100 microM, depending on the isoform, species and the presence of other cations. Calsequestrin monomers have a molecular mass of approximately 40 kDa and contain approximately 400 residues. The monomer contains three domains each with a compact alpha-helical/beta-sheet thioredoxin fold which is stable in the presence of Ca2+. The protein polymerises when Ca2+ concentrations approach 1mM. The polymer is anchored at one end to ryanodine receptor (RyR) Ca2+ release channels either via the intrinsic membrane proteins triadin and junctin or by binding directly to the RyR. It is becoming clear that calsequestrin has several functions in the lumen of the SR in addition to its well-recognised role as a Ca2+ buffer. Firstly, it is a luminal regulator of RyR activity. When triadin and junctin are present, calsequestrin maximally inhibits the Ca2+ release channel when the free Ca2+ concentration in the SR lumen is 1mM. The inhibition is relieved when the Ca2+ concentration alters, either because of small changes in the conformation of calsequestrin or its dissociation from the junctional face membrane. These changes in calsequestrin's association with the RyR amplify the direct effects of luminal Ca2+ concentration on RyR activity. In addition, calsequestrin activates purified RyRs lacking triadin and junctin. Further roles for calsequestrin are indicated by the kinase activity of the protein, its thioredoxin-like structure and its influence over store operated Ca2+ entry. Clearly, calsequestrin plays a major role in calcium homeostasis that extends well beyond its ability to buffer Ca2+ ions.     

The cDNA and deduced amino acid sequence of the gamma subunit of the L-type calcium channel from rabbit skeletal muscle

Complementary DNAs for the gamma subunit of the calcium channel of rabbit skeletal muscle were isolated on the basis of peptide sequences derived from the purified protein. The deduced primary structure is without homology to other known protein sequences and is consistent with the gamma subunit being an integral membrane protein.     

A combined non-denaturing and denaturing gel electrophoretic analysis of the subunit composition of a membrane protein: the skeletal muscle L-type calcium channel

Using a non-denaturing digitonin-based polyacrylamide gradient gel electrophoretic system we identified the dihydropyridine-sensitive Ca2+ channel from skeletal muscle as a high molecular weight protein of greater than 700 kDa. When this protein was excised from the native gels and re-electrophoresed into SDS gels, it dissociated into the alpha 1, alpha 2, beta, gamma and delta peptides previously suggested to be putative subunits of these Ca2+ channels. The stoichiometry of the alpha 1:alpha 2:beta:gamma peptides was (-)1:1:1:1. The presence of the alpha 1 and alpha 2 peptides in the high molecular weight native complex was directly demonstrated with anti-alpha 1 and anti-alpha 2 antibodies. The apparent specific association of the peptides was demonstrated by the finding that the previously separated alpha 1 and alpha 2 peptides did not co-migrate with the native complex in non-denaturing gels. The results of this previously untried analysis support the concept that the skeletal muscle Ca2+ channels are multisubunit proteins. The combined non-denaturing and denaturing gel analyses may be of general utility for the analysis of other membrane proteins.     

Gamma 1 subunit interactions within the skeletal muscle L-type voltage-gated calcium channels

Voltage-gated calcium channels mediate excitationcontraction coupling in the skeletal muscle. Their molecular composition, similar to neuronal channels, includes the pore-forming alpha(1) and auxiliary alpha(2)delta, beta, and gamma subunits. The gamma subunits are the least characterized, and their subunit interactions are unclear. The physiological importance of the neuronal gamma is emphasized by epileptic stargazer mice that lack gamma(2). In this study, we examined the molecular basis of interaction between skeletal gamma(1) and the calcium channel. Our data show that the alpha(1)1.1, beta(1a), and alpha(2)delta subunits are still associated in gamma(1) null mice. Reexpression of gamma(1) and gamma(2) showed that gamma(1), but not gamma(2), incorporates into gamma(1) null channels. By using chimeric constructs, we demonstrate that the first half of the gamma(1) subunit, including the first two transmembrane domains, is important for subunit interaction. Interestingly, this chimera also restores calcium conductance in gamma(1) null myotubes, indicating that the domain mediates both subunit interaction and current modulation. To determine the subunit of the channel that interacts with gamma(1), we examined the channel in muscular dysgenesis mice. Cosedimentation experiments showed that gamma(1) and alpha(2)delta are not associated. Moreover, alpha(1)1.1 and gamma(1) subunits form a complex in transiently transfected cells, indicating direct interaction between the gamma(1) and alpha(1)1.1 subunits. Our data demonstrate that the first half of gamma(1) subunit is required for association with the channel through alpha(1)1.1. Because subunit interactions are conserved, these studies have broad implications for gamma heterogeneity, function and subunit association with voltage-gated calcium channels.     

Dihydropyridine-sensitive calcium channels from skeletal muscle. I. Roles of subunits in channel activity.

Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are hetero-oligomeric proteins. Little is known about the functional roles of the various subunits, except that the alpha 1 subunit is the essential channel unit. We have reconstituted both partially purified holomeric channels and the separated subunits into liposomes and measured their properties using an assay based on the Ca2+ indicator dye fluo-3. The holomeric channels exhibited Ca2+ influx that was sensitive to membrane potential achieved by the addition of valinomycin in the presence of a K+ gradient. Dissipation of the K+ gradient resulted in the loss of the valinomycin-sensitive Ca2+ flux. In addition, the reconstituted channels were: 1) activated by the dihydropyridine Ca2+ channel activator Bay K 8644 in a dose-dependent manner with a Kd of 20 nM; 2) inhibited by various types of Ca2+ channel inhibitors including the dihydropyridine (+)-PN 200-110, the phenylalkylamine verapamil, and the benzothiazepine d-cis-diltiazem; and 3) modulated in a stereoselective manner by the enantiomers of the dihydropyridine S-202-791. The purified channels used in this work possessed an alpha 1 subunit of 165 kDa and did not appear to contain a larger alpha 1 subunit of approximately 210 kDa, suggesting that channel activity with properties similar to those observed in intact cells can be supported with an alpha 1 subunit of 165 kDa. Reconstituted channels that were 85% depleted in the alpha 2/delta subunits showed a significant decrease in the initial rate of Ca2+ influx induced by valinomycin, but retained responsiveness to Bay K 8644 and (+)-PN 200-110. When the separated alpha 2 and delta subunits were added back to the alpha 1 subunit-containing preparation, the channels exhibited their normal rate of Ca2+ influx. These results demonstrated that the dihydropyridine-sensitive Ca2+ channels from skeletal muscle require the presence of the alpha 2.gamma complex in stoichiometric amounts to exhibit full activity.     

Internal and external effects of dihydropyridines in the calcium channel of skeletal muscle

The agonist effect of the dihydropyridine (DHP) (-)Bay K 8644 and the inhibitory effects of nine antagonist DHPs were studied at a constant membrane potential of 0 mV in Ca channels of skeletal muscle transverse tubules incorporated into planar lipid bilayers. Four phenylalkylamines (verapamil, D600, D575, and D890) and d-cis-diltiazem were also tested. In Ca channels activated by 1 microM Bay K 8644, the antagonists nifedipine, nitrendipine, PN200-110, nimodipine, and pure enantiomer antagonists (+)nimodipine, (-)nimodipine, (+)Bay K 8644, inhibited activity in the concentration range of 10 nM to 10 microM. Effective doses (ED50) were 2 to 10 times higher when HDPs were added to the internal side than when added to the external side. This sidedness arises from different structure-activity relationships for DHPs on both sides of the Ca channel since the ranking potency of DHPs is PN200-110 greater than (-)nimodipine greater than nifedipine approximately S207-180 on the external side while PN200-110 greater than S207-180 greater than nifedipine approximately (-)nimodipine on the internal side. A comparison of ED50's for inhibition of single channels by DHPs added to the external side and ED50's for displacement of [3H]PN200-110 bound to the DHP receptor, revealed a good quantitative agreement. However, internal ED50's of channels were consistently higher than radioligand binding affinities by up to two orders of magnitude. Evidently, Ca channels of skeletal muscle are functionally coupled to two DHP receptor sites on opposite sides of the membrane.     

ADP-ribose stimulates the calcium release channel RyR1 in skeletal muscle of rat

The Ca(2+) mobilizing metabolite cyclic ADP-ribose has been shown to release Ca(2+) from intracellular ryanodine sensitive stores in many cells. However, the activation of the ryanodine receptor of skeletal muscle by cADP-ribose (cADPr) and its precursor and metabolite (beta-NAD(+) and ADPr) remains to be discussed. We studied the effect of ADPr on the Ca(2+) release channel of skeletal muscle RyR1 after incorporation of microsomes isolated from fast muscles of rat in planar lipid bilayers. We observed an increase in the electrophysiological activity of the channel after addition of ADPr (10 microM) at micromolar Ca(2+) concentrations, characterized by a time-lag. The increase in P(o) is mainly due to an increase in the open frequency. The long time course observed for the development of the ADPr effect may indicate that this activation induces a change in the conformation of the RyR1 channel, which increases its sensitivity to calcium.     

A calcium antagonist drug binding site in skeletal muscle sarcoplasmic reticulum: Evidence for a calcium channel

The sarcoplasmic reticulum (S.R.) of rabbit skeletal muscle has been found to contain a single, high affinity binding site for the Ca antagonist drug [³H] -nitrendipine. Two subfractions of the reticulum were studied, the heavy (HSR) and light (LSR) preparations, which exhibited similar nitrendipine equilibrium dissociation constants (K_D) of 1nM. Crude cardiac and brain membranes assayed under the same conditions exhibited K_D values of 0.2–0.3nM. The concentration of binding sites per mg. protein (B_max) in HSR was found to be very high, namely 6.7 picomoles/mg, some four times greater than that of LSR. [³H] -nitrendipine binding to HSR was reversible and inhibited by the Ca antagonists flunarizine and verapamil, and by the intracellular Ca release antagonist TMB-8 (8-diethylamino-octyl 3,4,5- trimethylbenzoate hydrochloride). However, unlabelled nitrendipine at 2 × 10^?5M had no effect on contraction of isolated electrically stimulated rabbit lumbrical or rat diaphragm muscles, nor did it affect the neuromuscular junction as studied in rat phrenic nerve-diaphragm preparations. Also, little effect of 2 × 10^?5M nitrendipine was seen on net ⁴⁵Ca uptake by HSR. These results suggest that [³H] -nitrendipine binding to skeletal muscle S.R. resembles that of brain membranes, which also contain a high affinity binding site for [³H] -nitrendipine and which similarly are pharmacologically insensitive to this dihydropyridine type of Ca channel blocking agent. Since HSR is also enriched in calsequestrin and terminal cysternae from which Ca is released in vivo, it seems likely that the [³H]- nitrendipine binding sites in S.R. are associated with Ca channels in the S.R.     

A long isoform of the epithelial sodium channel alpha subunit forms a highly active channel

A long isoform of the human Epithelial Sodium Channel (ENaC) α subunit has been identified, but little data exist regarding the properties or regulation of channels formed by α₇₂₈. The baseline whole cell conductance of oocytes expressing trimeric α₇₂₈βγ channels was 898.1 ± 277.2 and 49.59 ± 13.2 µS in low and high sodium solutions, respectively, and was 11 and 2 fold higher than the conductances of α₆₆₉βγ in same solutions. α₇₂₈βγ channels were also 2 to 5 fold less sensitive to activation by the serine proteases subtilisin and trypsin than α₆₆₉βγ in low and high Na⁺ conditions. The long isoform exhibited lower levels of full length and cleaved protein at the plasma membrane and a rightward shifted sensitivity to inhibition by increases of [Na⁺]_i. Both channels displayed similar single channel conductances of 4 pS, and both were activated to a similar extent by reducing temperature, altogether indicating that activation of baseline conductance of α₇₂₈βγ was likely mediated by enhanced channel activity or open probability. Expression of α₇₂₈ in native kidneys was validated in human urinary exosomes. These data demonstrate that the long isoform of αENaC forms the structural basis of a channel with different activity and regulation, which may not be easily distinguishable in native tissue, but may underlie sodium hyperabsorption and salt sensitive differences in humans.     

Hypochlorous acid modifies calcium release channel function from skeletal muscle sarcoplasmic reticulum.

We have previously demonstrated that H2O2 at millimolar concentrations induces Ca(2+) release from actively loaded sarcoplasmic reticulum (SR) vesicles and induces biphasic [(3)H]ryanodine binding behavior. Considering that hypochlorous acid (HOCl) is a related free radical and has been demonstrated to be a more effective oxidant of proteins, we evaluated the effects of HOCl on sarcoplasmic reticulum Ca(2+)-channel release mechanism. In a concentration-dependent manner, HOCl activates the SR Ca(2+) release channel and induces rapid release of Ca from actively loaded vesicles. HOCl-induced Ca(2+) release is inhibited in the presence of millimolar concentrations of DMSO. High-affinity [(3)H]ryanodine binding is also enhanced at concentrations from 10 to 100 microM. At HOCl concentrations of >100 microM, equilibrium binding is inhibited. HOCl stimulation of binding is inhibited by the addition of dithiothreitol. The direct interaction between HOCl and the Ca(2+) release mechanism was further demonstrated in single-channel reconstitution experiments. HOCl, at 20 microM, activated the Ca(2+) release channel after fusion of a SR vesicle to a bilayer lipid membrane. At 40 microM, Ca(2+)-channel activity was inhibited. Pretreatment of SR vesicles with HOCl inhibited the fluorescence development of a fluorogenic probe specific to thiol groups critical to channel function. These results suggest that HOCl at micromolar concentrations can modify SR Ca(2+) handling.