Comparison of regulated passive membrane conductance in action potential–firing fast- and slow-twitch muscle

In several pathological and experimental conditions, the passive membrane conductance of muscle fibers (G_m) and their excitability are inversely related. Despite this capacity of G_m to determine muscle excitability, its regulation in active muscle fibers is largely unexplored. In this issue, our previous study (Pedersen et al. 2009. J. Gen. Physiol. doi:10.1085/jgp.200910291) established a technique with which biphasic regulation of G_m in action potential (AP)-firing fast-twitch fibers of rat extensor digitorum longus muscles was identified and characterized with temporal resolution of seconds. This showed that AP firing initially reduced G_m via ClC-1 channel inhibition but after ∼1,800 APs, G_m rose substantially, causing AP excitation failure. This late increase of G_m reflected activation of ClC-1 and K_ATP channels. The present study has explored regulation of G_m in AP-firing slow-twitch fibers of soleus muscle and compared it to G_m dynamics in fast-twitch fibers. It further explored aspects of the cellular signaling that conveyed regulation of G_m in AP-firing fibers. Thus, in both fiber types, AP firing first triggered protein kinase C (PKC)-dependent ClC-1 channel inhibition that reduced G_m by ∼50%. Experiments with dantrolene showed that AP-triggered SR Ca²⁺ release activated this PKC-mediated ClC-1 channel inhibition that was associated with reduced rheobase current and improved function of depolarized muscles, indicating that the reduced G_m enhanced muscle fiber excitability. In fast-twitch fibers, the late rise in G_m was accelerated by glucose-free conditions, whereas it was postponed when intermittent resting periods were introduced during AP firing. Remarkably, elevation of G_m was never encountered in AP-firing slow-twitch fibers, even after 15,000 APs. These observations implicate metabolic depression in the elevation of G_m in AP-firing fast-twitch fibers. It is concluded that regulation of G_m is a general phenomenon in AP-firing muscle, and that differences in G_m regulation may contribute to the different phenotypes of fast- and slow-twitch muscle.     

Regulation of ClC-1 and KATP channels in action potential–firing fast-twitch muscle fibers

Action potential (AP) excitation requires a transient dominance of depolarizing membrane currents over the repolarizing membrane currents that stabilize the resting membrane potential. Such stabilizing currents, in turn, depend on passive membrane conductance (G_m), which in skeletal muscle fibers covers membrane conductances for K⁺ (G_K) and Cl⁻ (G_Cl). Myotonic disorders and studies with metabolically poisoned muscle have revealed capacities of G_K and G_Cl to inversely interfere with muscle excitability. However, whether regulation of G_K and G_Cl occur in AP-firing muscle under normal physiological conditions is unknown. This study establishes a technique that allows the determination of G_Cl and G_K with a temporal resolution of seconds in AP-firing muscle fibers. With this approach, we have identified and quantified a biphasic regulation of G_m in active fast-twitch extensor digitorum longus fibers of the rat. Thus, at the onset of AP firing, a reduction in G_Cl of ∼70% caused G_m to decline by ∼55% in a manner that is well described by a single exponential function characterized by a time constant of ∼200 APs (phase 1). When stimulation was continued beyond ∼1,800 APs, synchronized elevations in G_K (∼14-fold) and G_Cl (∼3-fold) caused G_m to rise sigmoidally to ∼400% of its level before AP firing (phase 2). Phase 2 was often associated with a failure to excite APs. When AP firing was ceased during phase 2, G_m recovered to its level before AP firing in ∼1 min. Experiments with glibenclamide (K_ATP channel inhibitor) and 9-anthracene carboxylic acid (ClC-1 Cl⁻ channel inhibitor) revealed that the decreased G_m during phase 1 reflected ClC-1 channel inhibition, whereas the massively elevated G_m during phase 2 reflected synchronized openings of ClC-1 and K_ATP channels. In conclusion, G_Cl and G_K are acutely regulated in AP-firing fast-twitch muscle fibers. Such regulation may contribute to the physiological control of excitability in active muscle.     

Absolute amounts and diffusibility of HSP72, HSP25, and αB-crystallin in fast- and slow-twitch skeletal muscle fibers of rat

Heat shock proteins (HSPs) are essential for normal cellular stress responses. Absolute amounts of HSP72, HSP25, and αB-crystallin in rat extensor digitorum longus (EDL) and soleus (SOL) muscle were ascertained by quantitative Western blotting to better understand their respective capabilities and limitations. HSP72 content of EDL and SOL muscle was only ～1.1 and 4.6 μmol/kg wet wt, respectively, and HSP25 content approximately twofold greater (～3.4 and ～8.9 μmol/kg, respectively). αB-crystallin content of EDL muscle was ～4.9 μmol/kg but in SOL muscle was ～30-fold higher (～140 μmol/kg). To examine fiber heterogeneity, HSP content was also assessed in individual fiber segments; every EDL type II fiber had less of each HSP than any SOL type I fiber, whereas the two SOL type II fibers examined were indistinguishable from the EDL type II fibers. Sarcolemma removal (fiber skinning) demonstrated that 10-20% of HSP25 and αB-crystallin was sarcolemma-associated in SOL fibers. HSP diffusibility was assessed from the extent and rate of diffusion out of skinned fiber segments. In unstressed SOL fibers, 70-90% of each HSP was readily diffusible, whereas ～95% remained tightly bound in fibers from SOL muscles heated to 45°C. Membrane disruption with Triton X-100 allowed dispersion of HSP72 and sarco(endo)plasmic reticulum Ca(2+)-ATPase pumps but did not alter binding of HSP25 or αB-crystallin. The amount of HSP72 in unstressed EDL muscle is much less than the number of its putative binding sites, whereas SOL type I fibers contain large amounts of αB-crystallin, suggesting its importance in normal cellular function without upregulation.     

An analysis of extracellular single muscle fibre action potential field —Modelling results

The radial variability of extracellular single muscle fibre action potential (SFAP) field was studied on the basis of an SFAP theoretical model originally constructed by Rosenfalck (1969). Amplitude and time parameters of the SFAP as a function of the radial distance r10 mm from the fibre were described by simple mathematical expressions. The results obtained were compared with existing experimental data from different authors and discussed in detail.     

Viscoelastic properties of passive skeletal muscle in compression—Cyclic behaviour

Skeletal muscle relaxation behaviour in compression has been previously reported, but the anisotropic behaviour at higher loading rates remains poorly understood. In this paper, uniaxial unconfined cyclic compression tests were performed on fresh porcine muscle samples at various fibre orientations to determine muscle viscoelastic behaviour. Mean compression level of 25% was applied and cycles of 2% and 10% amplitude were performed at 0.2–80 Hz. Under cycles of low frequency and amplitude, linear viscoelastic cyclic relaxation was observed. Fibre/cross-fibre results were qualitatively similar, but the cross-fibre direction was stiffer (ratio of 1.2). In higher amplitude tests nonlinear viscoelastic behaviour with a frequency dependent increase in the stress cycles amplitude was found (factor of 4.1 from 0.2 to 80 Hz).The predictive capability of an anisotropic quasi-linear viscoelastic model previously fitted to stress-relaxation data from similar tissue samples was investigated. Good qualitative results were obtained for low amplitude cycles but differences were observed in the stress cycle amplitudes (errors of 7.5% and 31.8%, respectively, in the fibre/cross-fibre directions). At higher amplitudes significant qualitative and quantitative differences were evident. A nonlinear model formulation was therefore developed which provided a good fit and predictions to high amplitude low frequency cyclic tests performed in the fibre/cross-fibre directions. However, this model gave a poorer fit to high frequency cyclic tests and to relaxation tests. Neither model adequately predicts the stiffness increase observed at frequencies above 5 Hz.Together with data previously presented, the experimental data presented here provide a unique dataset for validation of future constitutive models for skeletal muscle in compression.     

Mechanism of action of βadrenergic receptor blocking agents in angina pectoris: Comparison of action of propranolol with dexpropranolol and practolol

The effect on exercise tolerance of racemic propranolol has been assessed in eight angina pectoris patients and compared with that of dexpropranolol (the dextro isomer of propranolol), practolol (I.C.I. 50172), and saline. Dexpropranolol has the same local anaesthetic action as propranolol with negligible β-adrenergic receptor blocking activity, while practolol is a cardio-selective β-adrenergic blocking agent which does not have local anaesthetic activity.Saline and dexpropranolol had no significant effect on exercise time; racemic propranolol and practolol improved exercise tolerance in six subjects, the response to the two drugs being very similar in individual patients. It was concluded that the beneficial effect of propranolol in angina pectoris results from its action as a β-adrenergic receptor blocking agent and is not due to its local anaesthetic, or quinidine-like, activity.     

Evidence for HCO3− conductance pathways in nutrient membrane of bullfrog antrum

The effect of changing the nutrient-side HCO₃⁻ concentration on potential difference (PD) and resistance in bullfrog antrum bathing in Cl⁻ media was determined. Changes in HCO₃⁻ concentration were from 25 mM to several lower concentrations and back to 25mM. A plot of 6ΔPD6 versus log [HCO₃⁻] gave a linear relation for changes of HCO₃ concentration from 25 down to 3.1 mM and back to 25 mM but deviated to some extent for changes to 1.6 mM. In these experiments, changes from higher to lower HCO₃⁻ concentrations gave a less rapid initial PD response than those in the reverse direction. This result eliminated H⁺ conductance pathways as being predominant. Experiments were done in which in the first part changes were made in nutrient solution from 5% CO₂ and 25 mM HCO₃⁻ to 0.6% CO₂ and 3 mM HCO₃⁻ and in the second part the same changes with a simultaneous change of secretory solution from 5% to 10% CO₂. The magnitude of PD decrease was greater by 4.5 mV in the second part. This result indicated that HCO₃⁻ conductance pathways rather than OH⁻ conductance pathways predominated. There was no evidence of HCO₃⁻, OH⁻ and H⁺ conductance pathways in secretory membrane.     

Interpretation of relevance of sodium–calcium exchange in action potential of diabetic rat heart by mathematical model

Sarcolemmal Na⁺–Ca²⁺ exchange plays a central role in ion transport of the myocardium and the current carried with it contributes to the late phase of the action potential (AP) besides the contribution of outward K⁺-currents. In this study, the mathematical model for AP of the diabetic rat ventricular myocytes [34] was modified and used for the diabetic rat papillary muscle. We used our experimentally measured values of two K⁺-currents; transient outward current, I_to and steady-state outward current, I_ss, as well as L-type Ca²⁺-current, I_CaL, then compared with the simulated values. We have demonstrated that the prolongation in the AP of the papillary muscle of the diabetic rats are not due to the alteration of I_CaL but mainly due to the inhibition of the K⁺-currents and also the Na⁺–Ca²⁺ exchanger current, I_Na–Ca. In combination with our experimental data on sodium-selenite-treated diabetic rats, our simulation results provide new information concerning plausible ionic mechanisms, and second a possible positive effect of selenium treatment on the altered I_Na–Ca for the observed changes in the AP duration of streptozotocin-induced diabetic rat heart. (Mol Cell Biochem 269: 121–129, 2005)     

Changes of macromolecular organizations in nonjunctional sarcolemmas after cross-innervation──a study offast-and slow-twitch muscle fibres in rats

INTRODUCTIONMa1n11la1iaIlskeletal1llusclefibresare(listiIlgl1ishedint()severaltypesacc()rdillgtodifferentclassificatiollscl1e111es.Eachtypeofmusc1efibresl1asasetofspecificcharacteristics.Ollewell-kllowl1waytoclassifymusclefibresistodividethemintotwobroadtypestslow-twitcI1ortypeIfibres,andfast-twitchortypelIfibres.ThefOrmerllormallyappearsreda11dthelaterappearswhite.Theypossesswidedifferellceswithrespectt()physiol()gical,biochemical,andmorpho1ogica1pl1ellotypiccharacteristics,suchasisom…     

Control and role of pH in peptide–lipid interactions in oriented membrane samples

To understand the molecular mechanisms of amphiphilic membrane-active peptides, one needs to study their interactions with lipid bilayers under ambient conditions. However, it is difficult to control the pH of the sample in biophysical experiments that make use of mechanically aligned multilamellar membrane stacks on solid supports. HPLC-purified peptides tend to be acidic and can change the pH in the sample significantly. Here, we have systematically studied the influence of pH on the lipid interactions of the antimicrobial peptide PGLa embedded in oriented DMPC/DMPG bilayers. Using solid-state NMR (³¹P, ²H, ¹⁹F), both the lipid and peptide components were characterized independently, though in the same oriented samples under typical conditions of maximum hydration. The observed changes in lipid polymorphism were supported by DSC on multilamellar liposome suspensions. On this basis, we can present an optimized sample preparation protocol and discuss the challenges of performing solid-state NMR experiments under controlled pH. DMPC/DMPG bilayers show a significant up-field shift and broadening of the main lipid phase transition temperature when lowering the pH from 10.0 to 2.6. Both, strongly acidic and basic pH, cause a significant degree of lipid hydrolysis, which is exacerbated by the presence of PGLa. The characteristic re-alignment of PGLa from a surface-bound to a tilted state is not affected between pH of 7 to 4 in fluid bilayers. On the other hand, in gel-phase bilayers the peptide remains isotropically mobile under acidic conditions, displays various co-existing orientational states at pH 7, and adopts an unknown structural state at basic pH.     

Comparison of wild-type and UV-mutant β-glucanase-producing strains of Talaromyces emersonii with potential in brewing applications

A screen of 46 UV-mutant strains of the moderately thermophilic fungus Talaromyces emersonii yielded two mutants (TC2, TC5) that displayed gross morphological differences to the parent strain and enhanced activity against mixed linkage cereal -glucans. Activity against -(1, 3)(1, 4)-d-glucan from barley (BBGase) was measured during growth of the mutant and wild-type strains on a variety of carbon sources, ranging from solka floc to crude cereal fractions. In liquid culture, TC2 and TC5 secreted 1.2- to 8.6-fold more BBGase than the parent strain and markedly less -glucosidase (exo-activity); enzyme levels were dependent on the carbon source. Cellulose induced high BBGase. However, beet pulp, wheat bran, carob and tea-leaves were cheap and effective inducers. T. emersonii wild-type, TC2 and TC5 crude enzyme preparations achieved similar end-points during the hydrolysis of commercial barley -glucan (13.0–16.9%), but were more active against crude -glucan from barley (16.0–24.2% hydrolysis). The products of hydrolysis were quantified by high-performance anion-exchange chromatography. Mash trials indicated that enzyme preparations from all three organisms effected a significant reduction in wort viscosity and residual mash -glucan. Finally, TC2 and TC5 produce more efficient -glucan-depolymerizing enzymes; and wheat bran and solka floc can be used to provide inexpensive and potent enzyme cocktails with potential in brewing applications.     

Evidence for HCO3− conductance pathways in nutrient membrane of resting frog fundus

The effect on potential difference (PD) and resistance in Cl⁻ media bathing the resting fundus of Rana pipiens was determined for nutrient HCO₃⁻ changes from 25 mM to several lower concentrations and back to 25 mM. The graph of |vbΔPD|vb versus log[HCO₃⁻] was linear for changes from 25 down to 3.1 mM and also back to 25 mM, but deviated considerably for changes to 1.6 mM. The fact that changes from higher to lower HCO₃⁻ gave a less rapid initial PD response than the reverse direction eliminated H⁺ conductance pathways as being predominant. Experiments were done in which in the first part changes were made in the nutrient solution from 5% CO₂ and 25 mM HCO₃⁻ to 0.6% CO₂ and 3 mM HCO₃⁻ and in the second part, the same changes with the simultaneous change of secretory solution from 5% to 10% CO₂. The magnitude of the PD decrease was greater by 4.0 mV in the second part. This result indicated that HCO₃⁻ rather than OH⁻ conductance pathways predominated. On the secretory side, the change from 25 to 3.1 mM HCO₃⁻ gave a small but significant change in PD. The latter effect was too small to determine whether HCO₃⁻ pathways existed in the secretory membrane.     

Cell cycle–regulated membrane binding of NuMA contributes to efficient anaphase chromosome separation

Accurate and efficient separation of sister chromatids during anaphase is critical for faithful cell division. It has been proposed that cortical dynein–generated pulling forces on astral microtubules contribute to anaphase spindle elongation and chromosome separation. In mammalian cells, however, definitive evidence for the involvement of cortical dynein in chromosome separation is missing. It is believed that dynein is recruited and anchored at the cell cortex during mitosis by the α subunit of heterotrimeric G protein (Gα)/mammalian homologue of Drosophila Partner of Inscuteable/nuclear mitotic apparatus (NuMA) ternary complex. Here we uncover a Gα/LGN-independent lipid- and membrane-binding domain at the C-terminus of NuMA. We show that the membrane binding of NuMA is cell cycle regulated—it is inhibited during prophase and metaphase by cyclin-dependent kinase 1 (CDK1)–mediated phosphorylation and only occurs after anaphase onset when CDK1 activity is down-regulated. Further studies indicate that cell cycle–regulated membrane association of NuMA underlies anaphase-specific enhancement of cortical NuMA and dynein. By replacing endogenous NuMA with membrane-binding-deficient NuMA, we can specifically reduce the cortical accumulation of NuMA and dynein during anaphase and demonstrate that cortical NuMA and dynein contribute to efficient chromosome separation in mammalian cells.     

Has nonquantal acetylcholine secretion from motor nerve endings a role in neurotrophic control of resting membrane potential in rat muscle fibers?

The resting membrane potential of fibers of the rat diaphragm was measured by a microelectrode technique 3 h after division of the phrenic nerve and incubation in culture medium for 5 days after denervation. The membrane potential was recorded in synaptic regions of fibers close to (2–3 mm) and distant from (9–11 mm) the site of nerve division. The membrane potential of the synaptic region of the close fibers 3 h after denervation became smaller, whereas that of the synaptic region of distant fibers did not change relative to the control. Placing the muscle 3 h after denervation into medium with carbamylcholine (1·10^–8 M), cGMP (1·10^–4 M), or dibutyryl-cGMP (1·10^–6 M) led to hyperpolarization of the synaptic region of the close fibers but did not change the resting potential in the synaptic region of the distant fibers, and abolished differences between them. Five days after division of the nerve, incubation of the muscle in a solution with the above-mentioned substances did not affect the resting membrane potential. Nonquantal release of acetylcholine from motor nerve endings, assessed by the amplitude of hyperpolarization of the postsynaptic membrane, induced by application of curarine against the background of acetylcholine esterase inhibition, 3 h after denervation was identical in the synaptic region of the close and distant fibers and did not differ from the control. It is postulated that the postdenervation fall of membrane potential of rat muscle fibers is not due to disturbance of nonquantal secretion of acetylcholine from motor nerve endings.S. V. Kurashov Kazan' Medical Institute, Ministry of Health of the USSR. Translated from Neirofiziologiya, Vol. 17, No. 3, pp. 358–365, May–June, 1985.     

The effect of culture and membrane potential on Goα expression in neonatal rat cardiac myocytes

The effects of culture and membrane potential on G_o39 expression were examined in neonatal rat cardiac myocytes. During six days of culture, the amount of G_o39 in myocytes increased six-fold. The increase in G_o39 appeared to be programmed, since G_o39 of rat hearts also increased in vivo within three days after birth before declining by six days after birth. Furthermore, the age of the rat from which cardiac myocytes were isolated determined the amount of G_o39 that accumulated in cultured cells with myocytes from two day-old rats producing more G_o39 than myocytes from six day-old rats. In addition, agents which alter membrane potential (KCl and bupivacaine) inhibited the accumulation of G_o39 in cultured myocytes. In an attempt to identify the signaling pathway in which cardiac G_o39 is involved, muscarinic receptor-stimulated inositol phosphate production was examined, but was found to be comparable in myocytes that had six-fold differences in G_o39 content. Thus G_o39 does not appear to couple muscarinic receptors to phospholipase C in rat cardiac myocytes.     

Glycines: role in α-helical membrane protein structures and a potential indicator of native conformation

Among the growing number of membrane protein structures in the Protein Data Bank, there are many transmembrane domains that appear to be native-like; at the same time, there are others that appear to have less than complete native-like character. Hence, there is an increasing need for validation tools that distinguish native-like from non-native-like structures. Membrane mimetics used in protein structural characterizations differ in numerous physicochemical properties from native membranes and provide many opportunities for introducing non-native-like features into membrane protein structures. One possible approach for validating membrane protein structures is based on the use of glycine residues in transmembrane domains. Here, we have reviewed the membrane protein structure database and identified a set of benchmark proteins that appear to be native-like. In these structures, conserved glycine residues rarely face the lipid interstices, and many of them participate in close helix-helix packing. Glycine-based validation allowed the identification of non-native-like features in several membrane proteins and also shows the potential for verifying the native-like character for numerous other membrane protein structures.