Channels In Skeletal Muscle Research Articles

Photochemical localization of the semotiadil binding region within the cardiac Ca 2+ channel α1 subunit. Comparison with the skeletal muscle counterpart

We have previously identified the binding region of a new Ca 2+ antagonist semotiadil in the skeletal muscle Ca 2+ channel. To the same semotiadil derivatives, the cardiac counterpart showed distinct and different binding characteristics: semotiadil and its photoaffinity analog D51-4700 inhibited [ 3H]PN200-110 binding to cardiac membrane preparations with IC 50 values of 13–20 μM, which are 10 times higher than those in skeletal muscle. Hill slopes of the binding inhibition were 0.94–1.0 for the cardiac channels compared to 0.63–0.67 for the skeletal muscle channels. A possible explanation for the difference is that the semotiadil binding site is differently conferred in cardiac and skeletal muscle Ca 2+ channels. To reveal this within the primary structure, photoaffinity labeling of cardiac membranes was employed. [ 3H]D51-4700 was photoincorporated in several polypeptides but only the α1 subunit of the Ca 2+ channel was photolabeled in a specific manner. Antibody mapping of the [ 3H]D51-4700-labeled α1 subunit with several anti-peptide antibodies revealed that the labeled site was located solely in a peptide fragment between Cys 1461 and Lys 1529. This region encompasses the labeled site of skeletal muscle, but contains several non-identical amino acid residues, which may participate in expressing different binding characteristics between the two muscle type Ca 2+ channels.

Sodium channel fragments: contributions to voltage sensitivity and ion selectivity.

The peptide strategy was employed to resolve structure-function relationships in the voltage-dependent sodium channel Two families of motifs were studied: the four voltage sensors S4 extended with the short cytoplasmic linkers L45 and the four P-regions, between S5 and S6, each from the homologous domains of the electric eel sodium channel. Macroscopic conductance experiments conducted with synthetic S4L45s in neutral lipid planar bilayers pointed to a moderate voltage-sensitivity for repeat IV which has no proline, whereas S4L45 of repeats I and II (Pro 19) and especially of repeat III (Pro 14) were much more voltage-sensitive. The influence both of Pro and its position within the sequence was confirmed by comparing the human skeletal muscle channel isoform D4/S4 wild-type and the R4P analogue. Circular dichroism spectroscopy shows highest and lowest helicities for repeats IV and III. The conformational transition (from helix to extended, mainly beta forms), which occurs when the solvent dielectric constant increases, was broader with repeat III. These structural and functional correlates suggest alternative gating mechanisms. The different contributions of each repeat also have effects at the level of the main selectivity filter, which suggests self-recognition between the four P-regions is a key component of intact sodium channel selectivity. In addition, the P-region from domain III is significantly voltage-sensitive and molecular dynamics simulations show that the C-terminal part of P-regions is mainly helical whilst the N-terminus tends to unfold. Such specializations of the four domains both in gating and selectivity are independently confirmed in recent electrophysiological studies.

Mechanistic link between lidocaine block and inactivation probed by outer pore mutations in the rat μ1 skeletal muscle sodium channel

1. Mutations that disrupt Na+ channel fast inactivation attenuate lidocaine (lignocaine)-induced use dependence; however, the pharmacological role of slower inactivation processes remains unclear. In Xenopus oocytes, tryptophan substitution in the outer pore of the rat skeletal muscle channel (micro1-W402) alters partitioning among fast- and slow-inactivated states. We therefore examined the effects of W402 mutations on lidocaine block. 2. Recovery from inactivation exhibited three kinetic components (IF, fast; IM, intermediate; IS, slow). The effects of W402A and W402S on IF and IS differed, but both mutants (with or without beta1 subunit coexpression) decreased the amplitude of IM. In wild-type channels, lidocaine imposed a delayed recovery component with intermediate kinetics, and use-dependent block was attenuated in both W402A and W402S. 3. To examine the pharmacological role of IS relative to IM, drug-exposed beta1-coexpressed channels were subjected to 2 min depolarizations. Lidocaine had no effect on sodium current (INa) after a 1 s hyperpolarization interval that allowed recovery from IM but not IS, suggesting that lidocaine affinity for IS is low. 4. Both W402 mutations reduced occupancy of IM in drug-free conditions, and also induced resistance to use-dependent block. We propose that lidocaine-induced use dependence may involve an allosteric conformational change in the outer pore.

Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice.

ATP-sensitive K+ (KATP) channels regulate many cellular functions by linking cell metabolism to membrane potential. We have generated KATP channel-deficient mice by genetic disruption of Kir6.2, which forms the K+ ion-selective pore of the channel. The homozygous mice (Kir6.2(-/-)) lack KATP channel activity. Although the resting membrane potential and basal intracellular calcium concentrations ([Ca2+]i) of pancreatic beta cells in Kir6.2(-/-) are significantly higher than those in control mice (Kir6.2(+/+)), neither glucose at high concentrations nor the sulfonylurea tolbutamide elicits a rise in [Ca2+]i, and no significant insulin secretion in response to either glucose or tolbutamide is found in Kir6.2(-/-), as assessed by perifusion and batch incubation of pancreatic islets. Despite the defect in glucose-induced insulin secretion, Kir6.2(-/-) show only mild impairment in glucose tolerance. The glucose-lowering effect of insulin, as assessed by an insulin tolerance test, is increased significantly in Kir6.2(-/-), which could protect Kir6.2(-/-) from developing hyperglycemia. Our data indicate that the KATP channel in pancreatic beta cells is a key regulator of both glucose- and sulfonylurea-induced insulin secretion and suggest also that the KATP channel in skeletal muscle might be involved in insulin action.

Novel inhibition of contractility by wortmannin in skeletal muscle.

1. The effects of wortmannin and 2-(4-morpholinyl)-8-phenyl-1[4H]-benzopyran-4-one (LY294002), inhibitors of phosphatidylinositol 3-kinase, on the contractile responses of murine skeletal muscle were studied. Wortmannin (10-100 microM) suppressed twitch and tetanic contraction evoked by field stimulation of diaphragm without causing elevation of muscle tone. The inhibition was quasi-irreversible with IC50 approximately 15 microM. In contrast, LY294002 increased twitch responses and elevated muscle tone. 2. Wortmannin reversibly depressed the maximal slope of action potential upstroke by approximately 40% and inhibited the membrane depolarization and spontaneous burst of action potential induced by crotamine, a polypeptide toxin that activates the Na+ channel of skeletal muscle. 3. Wortmannin inhibited contractures evoked by high K+, ryanodine and caffeine, but potentiated the contracture induced by rapamycin, which binds to myoplasmic FK506 binding protein, an immunophilin closely associated with the ryanodine receptor. The contractures elicited by cardiotoxin, which disrupts the integrity of sarcolemma and thereby elevates 'myoplasmic' Ca2+ level, were suppressed only slightly. 4. In placed left atrium and ventricular strip, wortmannin and LY294002 produced a positive inotropic effect. 5. The results suggest that, in addition to depressing the Ca2+ mobilization from sarcoplasmic reticulum, wortmannin exerts a novel inhibitory action on the excitation-contraction coupling in skeletal muscle but not in cardiac muscle.

Electrophysiological study of chimeric sodium channels from heart and skeletal muscle.

The alpha-subunit cDNAs encoding voltage-sensitive sodium channels of human heart (hH1) and rat skeletal muscle (rSkM1) have been expressed in the tsA201 mammalian cell line, in which inactivation properties appear to be normal in contrast to Xenopus oocytes. A series of rSkM1/hH1 chimeric sodium channels has been evaluated to identify the domains of the alpha-subunits that are responsible for a set of electrophysiological differences between hH1 and rSkM1, namely, midpoints and slope factors of steady-state activation and inactivation, inactivation kinetics and recovery from inactivation kinetics and their voltage-dependence. The phenotype of chimeric channels in which each hH1 domain was successively introduced into a rSkM1 alpha-subunit framework confirmed the following conclusions. (i) The D4 and or/C-ter. are responsible for the slow inactivation of hH1 sodium channels. (ii) Concerning the other differences between rSkM1 and hH1: steady-state activation and inactivation, kinetics of recovery from inactivation, the phenotypes are determined probably by more than one domain of the alpha-subunit.

Slow Inactivation in Human Cardiac Sodium Channels

The available pool of sodium channels, and thus cell excitability, is regulated by both fast and slow inactivation. In cardiac tissue, the requirement for sustained firing of long-duration action potentials suggests that slow inactivation in cardiac sodium channels may differ from slow inactivation in skeletal muscle sodium channels. To test this hypothesis, we used the macropatch technique to characterize slow inactivation in human cardiac sodium channels heterologously expressed in Xenopus oocytes. Slow inactivation was isolated from fast inactivation kinetically (by selectively recovering channels from fast inactivation before measurement of slow inactivation) and structurally (by modification of fast inactivation by mutation of IFM1488QQQ). Time constants of slow inactivation in cardiac sodium channels were larger than previously reported for skeletal muscle sodium channels. In addition, steady-state slow inactivation was only 40% complete in cardiac sodium channels, compared to 80% in skeletal muscle channels. These results suggest that cardiac sodium channel slow inactivation is adapted for the sustained depolarizations found in normally functioning cardiac tissue. Complete slow inactivation in the fast inactivation modified IFM1488QQQ cardiac channel mutant suggests that this impairment of slow inactivation may result from an interaction between fast and slow inactivation.

Imperatoxin A induces subconductance states in Ca2+ release channels (ryanodine receptors) of cardiac and skeletal muscle.

Single-channel and [3H]ryanodine binding experiments were carried out to examine the effects of imperatoxin activator (IpTxa), a 33 amino acid peptide isolated from the venom of the African scorpion Pandinus imperator, on rabbit skeletal and canine cardiac muscle Ca2+ release channels (CRCs). Single channel currents from purified CRCs incorporated into planar lipid bilayers were recorded in 250 mM KCl media. Addition of IpTxa in nanomolar concentration to the cytosolic (cis) side, but not to the lumenal (trans) side, induced substates in both ryanodine receptor isoforms. The substates displayed a slightly rectifying current-voltage relationship. The chord conductance at -40 mV was approximately 43% of the full conductance, whereas it was approximately 28% at a holding potential of +40 mV. The substate formation by IpTxa was voltage and concentration dependent. Analysis of voltage and concentration dependence and kinetics of substate formation suggested that IpTxa reversibly binds to the CRC at a single site in the voltage drop across the channel. The rate constant for IpTxa binding to the skeletal muscle CRC increased e-fold per +53 mV and the rate constant of dissociation decreased e-fold per +25 mV applied holding potential. The effective valence of the reaction leading to the substate was approximately 1.5. The IpTxa binding site was calculated to be located at approximately 23% of the voltage drop from the cytosolic side. IpTxa induced substates in the ryanodine-modified skeletal CRC and increased or reduced [3H]ryanodine binding to sarcoplasmic reticulum vesicles depending on the level of channel activation. These results suggest that IpTxa induces subconductance states in skeletal and cardiac muscle Ca2+ release channels by binding to a single, cytosolically accessible site different from the ryanodine binding site.

Weakening of ion-channel interactions of Na+ and Li+ in acetylcholine-receptor channels of frog skeletal muscle with an increase in agonist concentration.

The possibility that increases in agonist concentration beyond threshold levels may force changes in the character of high-conductance open states of skeletal muscle nicotinic acetylcholine receptor channels (nAChR) was examined by seeing whether differences in several critical ionic properties of nAChR currents could be detected with changes in agonist level. Single- and bi-ionic whole-cell currents of Na+ and Li+ in voltage-clamped frog (Rana pipiens) muscle fibers were measured during local superfusion of endplates with carbamylcholine (carb) at concentrations of 54 microm (low-carb) and 270 microM (high-carb). Three ionic properties that would be affected by changes in the open-state configuration of channel subunits were tested. First, ion-saturation characteristics. Peak Na+ and Li+ currents in low-carb trials showed sublinear dependence on ion concentrations from 0 to 60 mM with Km values of 78 (Na+) and 49 (Li+) mM and a power function slope of 0. 75 on double-log plot. In contrast, the concentration dependence of Na+ and Li+ currents in high-carb tests was linear through the origin with a power function slope of 1.02. Second, Na+/Li+ selectivity. The ratio of peak Na+ and Li+ currents in low-carb tests varied from 1.86 to 2.28 for ion concentrations of from 20 to 60 mM [mean = 2.02 +/- 0.06 (SEM)] whereas the ratio for high-carb trials ranged from only 1.29 to 1.52 [mean = 1.42 +/- 0.40 (SEM)]. Third, competitive interactions of Na+ and Li+ currents. Equimolar mixtures of Na+ and Li+ in low-carb tests produced bi-ionic inward currents which were never larger than the single-ion Na+ current alone, but bi-ionic currents at the high-carb level were always greater than the single-ion Na+ current, approximating the sum of the single-ion Na+ and Li+ currents in most cases. The results are consistent with a decrease in ion-channel binding at the high-carb level and support the possibility of agonist-induced changes in the high-conductance open-state configuration of nAChR subunits which result in a weakening of constraints on cation movements through the channel.

Characterization of K(ATP) channels in intact mammalian skeletal muscle fibres.

1. The aim of this study was to characterize the K(ATP) channel of intact rat skeletal muscle (rat flexor digitorum brevis muscle). Changes in membrane currents were recorded with two-electrode voltage-clamp of whole fibres. 2. The K(ATP) channel openers, levcromakalim and pinacidil (10-400 microM), caused a concentration-dependent increase in whole-cell chord conductance (up to approximately 1.5 mScm(-2)). The activated current had a weak inwardly rectifying current-voltage relation, a reversal potential near E(K) and nanomolar sensitivity to glibenclamide--characteristic of a K(ATP) channel current. Concentration-effect analysis revealed that levcromakalim and pinacidil were not particularly potent (EC50 approximately 186 microM, approximately 30 microM, respectively), but diazoxide was completely inactive. 3. The ability of both classical K(ATP) channel inhibitors (glibenclamide, tolbutamide, glipizide and 5-hydroxydecanoic acid) and a number of structurally related glibenclamide analogues to antagonize the levcromakalim-induced current was determined. Glibenclamide was the most potent compound with an IC50 of approximately 5 nM. However, the non-sulphonylurea (but cardioactive) compound 5-hydroxydecanoic acid was inactive in this preparation. 4. Regression analysis showed that the glibenclamide analogues used have a similar rank order of potency to that observed previously in vascular smooth muscle and cerebral tissue. However, two compounds (glipizide and DK13) were found to have unexpectedly low potency in skeletal muscle. 5. These experiments revealed K(ATP) channels of skeletal muscle to be at least 10x more sensitive to glibenclamide than previously found; this may be because of the requirement for an intact intracellular environment for the full effect of sulphonylureas to be realised. Pharmacologically, K(ATP) channels of mammalian skeletal muscle appear to resemble most closely K(ATP) channels of cardiac myocytes.

Glycosphingolipids of skeletal muscle: II. Modulation of Ca 2+-flux in triad membranes by gangliosides

Membrane vesicles of rabbit skeletal muscle were prepared and separated by sucrose density gradient centrifugation. The fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M) as characterized by their specific marker enzymes, ligand binding, and ion flux activities. The distribution of neutral glycosphingolipids and gangliosides in these membrane preparations has been documented in the preceding paper (J. Müthing, U. Maurer, U. Neumann, B. Kniep, and S. Weber-Schürholz, Carbohydr. Res., (1988) 135–145). G M3(Neu5Ac) is the dominant ganglioside, neolacto-series gangliosides are moderately expressed and ganglio-series gangliosides were found in minor quantities, however, all showing different qualitative and quantitative membrane-type specific patterns. The voltage dependent Ca 2+-channels of skeletal muscle reside prevalently in the triad enriched membrane fractions deduced from highest binding capacity of 1,4-dihydropyridines. Calcium channel complexes of triads were reconstituted into unilamellar phospholipid vesicles of 400 nm defined size and the active 45Ca 2+-uptake into intravesicular space was measured after incorporation of muscle specific gangliosides into the outer vesicle lipid bilayer in parallel to control liposomes without gangliosides. G M3(Neu5Ac) strongly increased the uptake of 45Ca 2+ (+285%) whereas G M3(Neu5Gc) severely inhibited the ion flux (−61%). Neolacto-series gangliosides evoked miscellaneous effects upon 45Ca 2+-flux depending on isomeric sialic acid configuration, oligosaccharide size and fatty acid chain length of the ceramide portion. VI 3Neu5Ac– nLcOse 6Cer (C 24-fatty acid), IV 3Neu5Ac– nLcOse 4Cer (C 16-fatty acid) and IV 6Neu5Ac– nLcOse 4Cer (C 16-fatty acid) strongly enhanced the 45Ca 2+-flux (+208, +162, and +120%, respectively), whereas IV 3Neu5Ac– nLcOse 4Cer (C 24-fatty acid), VI 3Neu5Ac– nLcOse 6Cer (C 16-fatty acid) and IV 6Neu5Ac– nLcOse 4Cer (C 24-fatty acid) slightly reduced 45Ca 2+-flux (−3, −6, and −17%, respectively). Out of all gangliosides tested in this study, G M1 showed the strongest stimulatory effect (+327%). G D1a and G T1b gave rise to remarkable flux-stimulation of +283 and +255%, respectively, whereas G D1b exhibited only a slightly positive effect (+38%). This data suggest a functional role of gangliosides in subcellular muscle membranes giving strong evidence that gangliosides are capable of modulating the cytosolic calcium level of muscle, which regulates muscle contraction.

Imperatoxin A Induces Subconductance States in Ca 2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

Imperatoxin A Induces Subconductance States in Ca 2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

Expression of a single gene produces both forms of skeletal muscle cyclic nucleotide-gated channels.

Cyclic nucleotide-gated cation channels in skeletal muscle are responsible for insulin-activated sodium entry into this tissue (J. E. M. McGeoch and G. Guidotti. J. Biol. Chem. 267: 832-841, 1992). These channels have previously been isolated from rabbit skeletal muscle by 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) affinity chromatography, which separates them into two populations differing in nucleotide affinity [L. C. Santy and G. Guidotti. Am. J. Physiol. 271 (Endocrinol. Metab. 34): E1051-E1060, 1996]. In this study, a polymerase chain reaction approach was used to identify skeletal muscle cyclic nucleotide-gated channel cDNAs. Rabbit skeletal muscle expresses the same cyclic nucleotide-gated channel as rabbit aorta (M. Biel, W. Altenhofen, R. Hullin, J. Ludwig, M. Freichel, V. Flockerzi, N. Dascal, U. B. Kaupp, and F. Hofmann. FEBS Lett. 329: 134-138, 1993). The entire cDNA for this gene was cloned from rabbit skeletal muscle and an antiserum to this protein produced. Expression of this cDNA produces a 63-kDa protein with cyclic nucleotide-gated channel activity. A similarly sized immunoreactive protein is present in sarcolemma. Purification of the expressed channels reveals that this single gene produces both native skeletal muscle channel populations.

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca2+ channel activity by cAMP-dependent protein kinase.

Preferential phosphorylation of specific proteins by cAMP-dependent protein kinase (PKA) may be mediated in part by the anchoring of PKA to a family of A-kinase anchor proteins (AKAPs) positioned in close proximity to target proteins. This interaction is thought to depend on binding of the type II regulatory (RII) subunits to AKAPs and is essential for PKA-dependent modulation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor, the L-type Ca2+ channel, and the KCa channel. We hypothesized that the targeted disruption of the gene for the ubiquitously expressed RIIalpha subunit would reveal those tissues and signaling events that require anchored PKA. RIIalpha knockout mice appear normal and healthy. In adult skeletal muscle, RIalpha protein levels increased to partially compensate for the loss of RIIalpha. Nonetheless, a reduction in both catalytic (C) subunit protein levels and total kinase activity was observed. Surprisingly, the anchored PKA-dependent potentiation of the L-type Ca2+ channel in RIIalpha knockout skeletal muscle was unchanged compared with wild type although it was more sensitive to inhibitors of PKA-AKAP interactions. The C subunit colocalized with the L-type Ca2+ channel in transverse tubules in wild-type skeletal muscle and retained this localization in knockout muscle. The RIalpha subunit was shown to bind AKAPs, although with a 500-fold lower affinity than the RIIalpha subunit. The potentiation of the L-type Ca2+ channel in RIIalpha knockout mouse skeletal muscle suggests that, despite a lower affinity for AKAP binding, RIalpha is capable of physiologically relevant anchoring interactions.

Effector mechanism of adenosine in acute ischemic preconditioning of skeletal muscle against infarction.

We used adenosine A1 receptor agonist N6-1(phenyl-2R-isopropyl)-adenosine (PIA), A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and ATP-sensitive K+ (KATP) channel blockers sodium 5-hydroxydecanoate (5-HD) and glibenclamide (Glib), as probes to investigate the role and mechanism of adenosine in ischemic preconditioning (IPC) of noncontractile skeletal muscle against infarction, using the pig latissimus dorsi muscle flap model. Except for Glib, all drugs were delivered to each muscle flap by 10-min local intra-arterial infusion to avoid systemic effects. Muscle flaps that were subjected to 4 h of global ischemia and 48 h of reperfusion sustained 40 +/- 2% infarction. IPC with three cycles of 10 min ischemia and reperfusion, preischemic adenosine, or PIA treatment reduced (P < 0.05) muscle infarction to 24 +/- 2, 18 +/- 2, and 24 +/- 2%, respectively. The anti-infarction effect of IPC and adenosine was blocked by DPCPX, 5-HD, and Glib (P < 0.05). Preischemic adenosine treatment also maintained higher muscle contents of phosphocreatine, ATP, and energy charge potential and lower muscle contents of dephosphorylated metabolites and lactate during ischemia and a lower muscle myeloperoxidase (MPO) activity during reperfusion compared with the control (P < 0.05). Preischemic adenosine treatment did not increase muscle content of adenosine during ischemia or reperfusion. Furthermore, adenosine given at the onset of reperfusion was not effective in attenuating muscle MPO activity or infarction. Taken together, these observations indicate that adenosine, through A1 receptors, initiates the mechanism of IPC with postreceptor involvement of KATP channels in skeletal muscle. However, adenosine is unlikely to play a key role in the effector mechanism. Presently, the cause and role of energy sparing and neutrophil inhibitory effects associated with the anti-infarction effect of preischemic adenosine treatment are unknown.

Interaction of S100A1 with the Ca2+ Release Channel (Ryanodine Receptor) of Skeletal Muscle

In the present report we studied the interaction between the skeletal muscle ryanodine receptor and the ubiquitous S100A1 Ca2+ binding protein. S100A1 did not affect equilibrium [3H]ryanodine binding to purified rabbit skeletal muscle terminal cisternae at 100 microM free [Ca2+]. At nanomolar free [Ca2+], however, S100A1 activated by 40 +/- 6.7% (mean +/- SE, n = 5) the [3H]ryanodine binding activity; the half-maximal concentration for stimulation of [3H]ryanodine binding was approximately 70 nM, a value well below the estimated S100A1 concentration in skeletal muscle fibers. Scatchard analysis of [3H]ryanodine binding performed in the presence of 100 microM EGTA indicates that S100A1 increases the apparent affinity of the receptor for ryanodine (Kd = 191 vs 383 nM in the presence and in the absence of 100 nM S100A1, respectively). The effect of S100A1 was also tested on the single-channel gating properties of the purified ryanodine receptor after reconstitution into a lipid planar bilayer. Currents carried by purified ryanodine receptor channels were modulated by both cis Ca2+ and ruthenium red. In the presence of nanomolar [Ca2+], S100A1 activated the channel by increasing (6.0 +/- 2.8)-fold (mean +/- SE, n = 3) the normalized open probability. The interaction between S100A1 and the purified RYR was verified using the optical biosensor BIAcore: we show that the two proteins interact directly both at millimolar and at nanomolar calcium concentrations. We next mapped the regions of the skeletal muscle RYR involved in the interaction with S100A1 by performing ligand overlays on a panel RYR of fusion proteins in the presence of 100 nM S100A1. Our results indicate that the skeletal muscle RYR contains three potential S100A1 binding domains. Binding of S100A1 to the RYR fusion proteins occurred at both nanomolar and millimolar free [Ca2+]. S100A1 binding domain 1 binds the ligand in the presence of 1 mM free [Ca2+] or 1 mM EGTA. Maximal binding to S100A1#2 was achieved in the presence of 1 mM free [Ca2+]. The S100A1#3 domain, which overlaps with calcium-dependent calmodulin binding domain 3 (CaM 3), exhibits weak and strong S100A1 binding activity in the presence of either millimolar or nanomolar Ca2+, respectively. The interaction between S100A1 and the purified RYR complex was also investigated by affinity chromatography: in the presence of nanomolar Ca2+, we observed binding of native RYR complex to S100A1-conjugated Sepharose. This interaction could be inhibited by the presence of RYR polypeptides encompassing S100A1 binding sites S100A1#1, S100A1#2, and S100A1#3.

Gating of the skeletal calcium release channel by ATP is inhibited by protein phosphatase 1 but not by Mg 2+

We have previously found that dephosphorylation/phosphorylation of the calcium release channel (CRC) of skeletal muscle confers channel sensitivity/insensitivity to the block by physiological [Mg 2+] (∼1 mM). These studies have now been extended to modulation by ATP. Terminal cisternae vesicles of sarcoplasmic reticulum were incorporated into planar lipid bilayers. CRC gating by ATP (0.67 mM), in the absence of Ca 2+ (< 1 nM), was studied by treatment with protein kinase A (PKA) or phosphatase 1 (PPT1) and assayed in the presence and absence of free Mg 2+ (1 mM). PPT1, PKA, and Mg 2+ were directly applied to the bilayer using the microsyringe method, which controls the environment of the CRC in the bilayer for phosphorylation/ dephosphorylation cycles and for assays. PKA treated channels were activated by ATP to high open probabilities, while PPT1 treated channels were not activatable by ATP. Opening and closing of channels during cycles of PKA and PPT1 applications, respectively, provided evidence that the change of CRC activity is due to cyclic phosphorylation/dephosphorylation. Free Mg 2+ (1 mM) did not block channels activated by ATP. The new finding is that channel gating by ATP can be controlled by the state of phosphorylation without inhibition by free Mg 2+.

Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons.

Membrane excitability in different tissues is due, in large part, to the selective expression of distinct genes encoding the voltage-dependent sodium channel. Although the predominant sodium channels in brain, skeletal muscle, and cardiac muscle have been identified, the major sodium channel types responsible for excitability within the peripheral nervous system have remained elusive. We now describe the deduced primary structure of a sodium channel, peripheral nerve type 1 (PN1), which is expressed at high levels throughout the peripheral nervous system and is targeted to nerve terminals of cultured dorsal root ganglion neurons. Studies using cultured PC12 cells indicate that both expression and targeting of PN1 is induced by treatment of the cells with nerve growth factor. The preferential localization suggests that the PN1 sodium channel plays a specific role in nerve excitability.

Kinetic analysis of the inhibitory effect of glibenclamide on KATP channels of mammalian skeletal muscle.

We investigated the block of KATP channels by glibenclamide in inside-out membrane patches of rat flexor digitorum brevis muscle. (1) We found that glibenclamide inhibited KATP channels with an apparent Ki of 63 nm and a Hill coefficient of 0.85. The inhibition of KATP channels by glibenclamide was unaffected by internal Mg2+. (2) Glibenclamide altered all kinetic parameters measured; mean open time and burst length were reduced, whereas mean closed time was increased.(3) By making the assumption that binding of glibenclamide to the sulphonylurea receptor (SUR) leads to channel closure, we have used the relation between mean open time, glibenclamide concentration and KD to estimate binding and unbinding rate constants. We found an apparent rate constant for glibenclamide binding of 9.9 x 10(7) m-1 sec-1 and an unbinding rate of 6.26 sec-1. (4) Glibenclamide is a lipophilic molecule and is likely to act on sulfonylurea receptors from within the hydrophobic phase of the cell membrane. The glibenclamide concentration within this phase will be greater than that in the aqueous solution and we have taken this into account to estimate a true binding rate constant of 1.66 x 10(6) m-1 sec-1.

Chloride-dependent sarcoplasmic reticulum Ca2+ release correlates with increased Ca2+ activation of ryanodine receptors

The mechanism by which chloride increases sarcoplasmic reticulum (SR) Ca2+ permeability was investigated. In the presence of 3 microM Ca2+, Ca2+ release from 45Ca(2+)-loaded SR vesicles prepared from procine skeletal muscle was increased approximately 4-fold when the media contained 150 mM chloride versus 150 mM propionate, whereas in the presence of 30 nM Ca2+, Ca2+ release was similar in the chloride- and the propionate-containing media. Ca(2+)-activated [3H]ryanodine binding to skeletal muscle SR was also increased (2- to 10-fold) in media in which propionate or other organic anions were replaced with chloride; however, chloride had little or no effect on cardiac muscle SR 45Ca2+ release or [3H]ryanodine binding. Ca(2+)-activated [3H]ryanodine binding was increased approximately 4.5-fold after reconstitution of skeletal muscle RYR protein into liposomes, and [3H]ryanodine binding to reconstituted RYR protein was similar in chloride- and propionate-containing media, suggesting that the sensitivity of the RYR protein to changes in the anionic composition of the media may be diminished upon reconstitution. Together, our results demonstrate a close correlation between chloride-dependent increases in SR Ca2+ permeability and increased Ca2+ activation of skeletal muscle RYR channels. We postulate that media containing supraphysiological concentrations of chloride or other inorganic anions may enhance skeletal muscle RYR activity by favoring a conformational state of the channel that exhibits increased activation by Ca2+ in comparison to the Ca2+ activation exhibited by this channel in native membranes in the presence of physiological chloride (< or = 10 mM). Transitions to this putative Ca(2+)-activatable state may thus provide a mechanism for controlling the activation of RYR channels in skeletal muscle.