Effects of the Intracellular Ca Ion Concentration upon the Excitability of the Muscle Fiber Membrane of a Barnacle

The membrane excitability and contraction were examined in single barnacle muscle fibers with different internal Ca⁺⁺ concentrations by using buffer solutions made up with EGTA and Ca-gluconate in various proportions. During the passage of dc currents the membrane shows all-or-none spike potentials for internal Ca⁺⁺ concentrations below about 8 x 10^-8 M, oscillatory potential changes in the range between 8 x 10^-8 to 5 x 10^-7 M, but neither oscillatory nor spike potentials were seen for concentrations above 5 x 10^-7 M. All-or-none spike potentials were suppressed when the internal Mg⁺⁺ concentration exceeded 5 mM. The suppression threshold of the internal Ca⁺⁺ concentration for the Sr spike is much higher than that for the Ca spike. The threshold concentration of internal Ca⁺⁺ for contraction was about 8 x 10^-7 M.     

Effect of External and Internal pH Changes on K and Cl Conductances in the Muscle Fiber Membrane of a Giant Barnacle

The membrane potential and conductance of the giant muscle fiber of a barnacle (Balanus nubilus Darwin) were analyzed in relation to changes in the external (3.5–10.0) and the internal (4.7–9.6) pH, under various experimental conditions. A sharp increase in membrane conductance, associated with a large increase in conductance to Cl ions, was observed when the external pH was lowered to values below 5.0. The ratio of Cl to K conductance in normal barnacle saline is between –1/7 at pH 7.7, whereas at pH 4.0 the ratio is about 6–9. The behavior of the membrane in response to pH changes in a Cl-depleted muscle fiber shows that the K conductance decreases with decreasing external pH for the whole range of pH examined. A steep increase in Cl conductance is also observed when the internal pH of the fiber is lowered below 5.0. The K to Cl conductance ratio increases with increasing internal pH in a manner very similar to that found when the external pH is raised above 5.0. These facts suggest that the membrane is amphoteric with positive and negative fixed charge groups having dissociation constants such that at pH greater than 5, negative groups predominate and cations permeate more easily than anions, while at lower pH positive groups predominate, facilitating the passage of anions through the membrane.     

Membrane Currents Carried by Ca, Sr, and Ba in Barnacle Muscle Fiber During Voltage Clamp

Membrane currents associated with voltage clamp of the giant muscle fibers of a barnacle, Balanus nubilus, were analyzed in terms of currents of the Ca and K channels. Although the activation of the K channel occurs more slowly than that of the Ca channel, both currents show a significant temporal overlap. The currents carried by Ca⁺⁺, Sr⁺⁺, or Ba⁺⁺ through the Ca channel were compared under the conditions at which this overlap was the least. When only one divalent cation is present in the solution, Ba⁺⁺ carries more current than Ca⁺⁺ or Sr⁺⁺ and the sequence of the current is Ba > Sr ≈ Ca. When the external solution contains a relatively high concentration of Co⁺⁺, which is a blocking agent for the Ca channel, inversion of the sequence occurs, to Ca > Sr > Ba. This is due to the fact that the blocking effect differs depending on which ion carries current through the Ca channel. The Ba current is most sensitive and the Ca current is least affected. Ba suppresses the current of the K channel, independently of its current-carrying function through the Ca channel.     

Relation between Membrane Potential Changes and Tension in Barnacle Muscle Fibers

Constant current pulses have been applied to single muscle fibers of the barnacle, Balanus nubilus Darwin, with an axial metal electrode. The membrane potential change, which took place over a large part of the muscle fiber, was measured with a similar electrode. Depolarizing pulses, if the voltage was greater than threshold, produced tension. The size of the tension was a function of the magnitude and the duration of the depolarizing pulses. The latency between the onset of depolarization and tension can be only in part attributable to mechanical factors. AC stimulation produced tension, but 5 to 10 seconds were required for the steady-state level of the tension to be reached. Muscles were depolarized in elevated K and studied after the contracture had terminated. If not too depolarized, further depolarization produced tension. Termination of hyperpolarizing pulses also produced tension, which decayed quite slowly. Hyperpolarizing pulses reduced, or abolished, any preexisting tension. Thus, it appears that at certain values of the membrane potential tension is set up, but there is also a slow process of accommodation present.     

Solvent Water for Electrolytes in the Muscle Fiber of the Giant Barnacle

Seven experiments are described which permit estimation of the "solvent water" or the "osmotically active water" of the dissected fiber from the giant barnacle, Balanus nubilus. Each of the first four experiments includes the measurement of a free ion activity in the myoplasm by means of a Na⁺, K⁺, or Cl^- ion-specific microelectrode. The fifth experiment makes use of a membrane potential vs. [K]_o curve. The last two experiments measured fiber water and fiber volume as bath osmolarity was changed. The seven independent estimations of solvent water ranged from 0.64 to 0.72 of fiber water with a mean of 0.68. Since the extracellular space of single fibers was about 7% of fiber water, it was concluded that 25% of analyzable water was not acting as solvent for the osmotically active solutes in the myoplasm.     

The Effects of Various Ions on Resting and Spike Potentials of Barnacle Muscle Fibers

Effects of monovalent cations and some anions on the electrical properties of the barnacle muscle fiber membrane were studied when the intra- or extracellular concentrations of those ions were altered by longitudinal intra-cellular injection. The resting potential of the normal fiber decreases linearly with increase of logarithm of [K⁺]_out and the decrement for a tenfold increase in [K⁺]_out is 58 mv when the product, [K⁺]_out ·[Cl^-]_out, is kept constant. It also decreases with decreasing [K⁺]_in but is always less than expected theoretically. The deviation becomes larger as [K⁺]_in increases and the resting potential finally starts to decrease with increasing [K⁺]_in for [K⁺]_in > 250 mM. When the internal K⁺ concentration is decreased the overshoot of the spike potential increases and the time course of the spike potential becomes more prolonged. In substituting for the internal K⁺, Na⁺ and sucrose affect the resting and spike potentials similarly. Some organic cations (guanidine, choline, tris, and TMA) behave like sucrose while some other organic cations (TEA, TPA, and TBA) have a specific effect and prolong the spike potential if they are applied intracellularly or extracellularly. In all cases the active membrane potential increases linearly with the logarithm of [Ca⁺⁺]_out/[K⁺]_in and the increment is about 29 mv for tenfold increase in this ratio. The fiber membrane is permeable to Cl^- and other smaller anions (Br^- and I^-) but not to acetate^- and larger anions (citrate^-, sulfate^-, and methanesulfonate^-).     

Electrically Silent Divalent Cation Entries in Resting and Active Voltage-Controlled Muscle Fibers

Ca²⁺ is known to enter skeletal muscle at rest and during activity. Except for the well-characterized Ca²⁺ entry through L-type channels, pathways involved in these Ca²⁺ entries remain elusive in adult muscle. This study investigates Ca²⁺ influx at rest and during activity using the method of Mn²⁺ quenching of fura-2 fluorescence on voltage-controlled adult skeletal muscle cells. Resting rate of Mn²⁺ influx depended on external [Mn²⁺] and membrane potential. At −80 mV, replacement of Mg²⁺ by Mn²⁺ gave rise to an outward current associated with an increase in cell input resistance. Calibration of fura-2 response indicated that Mn²⁺ influx was too small to be resolved as a macroscopic current. Partial depletion of the sarcoplasmic reticulum induced by a train of action potentials in the presence of cyclopiazonic acid led to a slight increase in resting Mn²⁺ influx but no change in cell input resistance and membrane potential. Trains of action potentials considerably increased Mn²⁺ entry through an electrically silent pathway independent of L-type channels, which provided 24% of the global Mn²⁺ influx at +30 mV under voltage-clamp conditions. Within this context, the nature and the physiological role of the Ca²⁺ pathways involved during muscle excitation still remain open questions.     

Membrane Characteristics of the Canine Papillary Muscle Fiber

Passive and active responses to intracellular and extracellular stimulation were studied in the canine papillary muscle. The electrotonic potential produced by extracellular polarization with the partition chamber method fitted the time course and the spatial decay expected from the cable theory (the time constant, 3.3 msec; the space constant, 1.2 mm). Contrariwise, spatial decay of the electrotonic potentials produced by intracellular polarization was very short and did not fit the decay curve expected for a simple cable, although only a small difference of time course in the electrotonic potentials produced by intracellular and extracellular polarizations was observed. A similar time course might result from the fact that when current flow results from intracellular polarization, the input resistance is less dependent on the membrane resistance. The foot of the propagated action potential rose exponentially with a time constant of 1.1 msec and a conduction velocity of 0.68 m/sec. The membrane capacity was calculated from the time constant of the foot potential and the conduction velocity to be 0.76 µF/cm². The responses of the papillary muscle membrane to intracellular stimulation differed from those to extracellular stimulation applied with the partition method in the following ways: higher threshold potential, shorter latency for the active response, linearity of the current-voltage relationship, and no reduction in the membrane resistance at the crest of the action potential during current flow.     

Membrane Potentials and Ion Permeability in a Cation Exchange Membrane

The nonequilibrium electrical potentials across an artificial membrane bathed by solutions of a single salt have been measured and calculated using the Goldman-Hodgkin-Katz equation and the irreversible thermodynamic equation. The latter equation predicts the observed potential differences over a 2500-fold concentration range, while application of a modified Goldman-Hodgkin-Katz equation leads to difficulties.     

Dynamic Properties of Giant Muscle Fibers of the Barnacle

Giant muscle fibers of the barnacle give graded, relativelyslow contractions, A plateau level, termed the unit response,occurs with stimuli of 3 msec, but pulses longer than 10–15msec give much greater tension or shortening. Repetitive stimulationwith pulses of 3 msec leads to a tetanus. The magnitude of activestate was determined, and found to be also graded in natureand slow to develop, though early in onset. The full developmentof active state requires 80–120 msec. A high level ofeffective series-elasticity was associated with the sarcomeresthemselves.     

The Impact of Magnesium on Isometric Twitch Parameters and Resting Membrane Potential of the Skeletal Muscle in Diabetic Rats

To present the relationship between oral magnesium supplementation, blood glucose, and changes in isometric twitch parameters, resting membrane potential (RMP), in the gastrocnemius muscle in diabetic rats. Sixty rats were used in this study. The rats were divided into four groups: control (drinking tap water, Group I, n = 15), control with treated with magnesium sulfate (10 g/L) (Group II, n = 15), diabetic (Group III, n = 15), and diabetic with treated with magnesium sulfate (10 g/L) (Group IV, n = 15). In Group II and IV, the level of plasma magnesium was increased comparing to those of the control group (p < 0.05). Isometric twitch tensions were decreased significantly in the Group III, but Group IV isometric twitch tensions were increased significantly. Group IV RMP values were close to the Group I. Hyperglycemia decreases gastrocnemius muscle isometric twitch tension and increases RMP in diabetic rats. Magnesium treatment can prevent these diabetic complications.     

A Calcium Aspect of the Effect of Noradrenaline on the Resting Membrane Potential in Somatic Muscle Cells of the Earthworm

A hyperpolarizing effect of noradrenaline (NA) on muscle cells of the earthworm caused by activation of the membrane ion pumps is eliminated in a Ca-free medium, in the case of replacement of Na⁺ by Mn²⁺, or when verapamil or chlorpromazine have been added to the bath solution. A decrease or an increase in the Ca²⁺ concentration in the solution, as well as caffeine application, do not influence the resting membrane potential (RMP) of muscle cells. It is supposed that signal transmission from the membrane adrenoreceptors to the ion pump of earthworm muscle cells by NA is provided via entry of extracellular Ca²⁺ ions into the cell with subsequent involving of Ca²⁺ acceptor proteins similar to calmodulin in vertebrate animals.     

The Structure of Mytilus Smooth Muscle and the Electrical Constants of the Resting Muscle

The individual muscle fibers of the anterior byssus retractor muscle (ABRM) of Mytilus edulis L. are uninucleate, 1.2–1.8 mm in length, 5 µm in diameter, and organized into bundles 100–200 µm in diameter, surrounded by connective tissue. Some bundles run the length of the whole muscle. Adjacent muscle cell membranes are interconnected by nexuses at frequent intervals. Specialized attachments exist between muscle fibers and connective tissue. Electrical constants of the resting muscle membrane were measured with intracellular recording electrodes and both extracellular and intracellular current-passing electrodes. With an intracellular current-passing electrode, the time constant τ, was 4.3 ± 1.5 ms. With current delivered via an extracellular electrode τ was 68.3 ± 15 ms. The space constant, λ, was 1.8 mm ± 0.4. The membrane input resistance, R_eff, ranged from 23 to 51 MΩ. The observations that values of τ depend on the method of passing current, and that the value of λ is large relative to fiber length and diameter are considered evidence that the individual muscle fibers are electrically interconnected within bundles in a three-dimensional network. Estimations are made of the membrane resistance, R_m, to compare the values to fast and slow striated muscle fibers and mammalian smooth muscles. The implications of this study in reinterpreting previous mechanical and electrical studies are discussed.     

Cation Permeability and Selectivity of a Root Plasma Membrane Calcium Channel

Calcium channels in the plasma membrane of root cells fulfill both nutritional and signaling roles. The permeability of these channels to different cations determines the magnitude of their cation conductances, their effects on cell membrane potential and their contribution to cation toxicities. The selectivity of the rca channel, a Ca²⁺-permeable channel from the plasma membrane of wheat (Triticum aestivum L.) roots, was studied following its incorporation into planar lipid bilayers. The permeation of K⁺, Na⁺, Ca²⁺ and Mg²⁺ through the pore of the rca channel was modeled. It was assumed that cations permeated in single file through a pore with three energy barriers and two ion-binding sites. Differences in permeation between divalent and monovalent cations were attributed largely to the affinity of the ion binding sites. The model suggested that significant negative surface charge was present in the vestibules to the pore and that the pore could accommodate two cations simultaneously, which repelled each other strongly. The pore structure of the rca channel appeared to differ from that of L-type calcium channels from animal cell membranes since its ion binding sites had a lower affinity for divalent cations. The model adequately accounted for the diverse permeation phenomena observed for the rca channel. It described the apparent submillimolar K _m for the relationship between unitary conductance and Ca²⁺ activity, the differences in selectivity sequences obtained from measurements of conductance and permeability ratios, the changes in relative cation permeabilities with solution ionic composition, and the complex effects of Ca²⁺ on K⁺ and Na⁺ currents through the channel. Having established the adequacy of the model, it was used to predict the unitary currents that would be observed under the ionic conditions employed in patch-clamp experiments and to demonstrate the high selectivity of the rca channel for Ca²⁺ influx under physiological conditions. Received: 23 August 1999/Revised: 12 November 1999     

Membrane Depolarization and the Metabolism of Muscle

The respiration of frog twitch muscles rises markedly when [K]₀israised; respiration is stimulated by levels of [K]₀ below thethreshold for contracture(Fenn, 1931).Respiration is also stimulatedby elevated [Rb]₀ and [Cs]₀ in direct relation to their abilityto depolarize the membrane. Respiration is stimulated even whenthe anions in the high [K]_o solution cannot permeate the membrane.If[K]₀is raised to 25 mM there is an increase in respiration whichis sustained for hours. If [K]₀is 30 mM or above, there is atransitory burst of stimulated respiration followed by a declineback to the basal level. The response to elevated[K]₀ can beblocked by divalent cations or by local anesthetics; the blockingagents act rapidly, probably on the cell membrane. Either extracellularcalcium or strontium is needed for a prolonged stimulation ofrespiration. Depolarization seems to increase respiration bycausing the release of calcium into the sarcoplasm. Since respirationis increased by a depolarization below the threshold for producinga contracture, respiration is a sensitive indicator of the sarcoplasmicconcentration of calcium. A model for the relation between sarcoplasmic[Ca] and membrane potential is proposed. Calcium can be releasedfrom a store in the cell, the released Ca⁺⁺ entering the sarcoplasm.The store is replenished by Ca⁺⁺ entering the fiber from theextracellular solution. When the membrane is depolarized, therate of release of calcium in the store is increased; at thesame time the rate at which extracellular calcium can replenishthe store is decreased.This model accounts well for the dataon respiration and also for the contractures of single musclefibers. Calcium probably acts within the cell to activate anATP-ase which causes an increase in ADP and hence an increasein respiration. Other investigators have found changes in theactivity of certain enzymes and in the permeability of the membrane;possibly these changes are also a direct response to an increasein sarcoplasmic calcium.     

Role of Cation and Anion Uptake in Salt-stimulated Elongation of Lettuce Hypocotyl Sections

The role of cation and anion uptake in salt-stimulated growth of light-grown, GA₃-treated lettuce (Lactuca sativa L.) hypocotyl sections was investigated. Potassium chloride (10 mm) causes a 2-fold increase in the growth rate of GA₃-treated hypocotyl sections without affecting the growth rate of sections incubated in the absence of GA₃. Salt uptake is the same in both treatments, and furthermore the uptake of cation and anion is stoichiometric during the first 24 hours under all incubation conditions. The importance of the anion for cation uptake is demonstrated in experiments with benzenesulfonate⁻ and iminodiacetate²⁻. When K⁺ and Na⁺ are supplied only as the benzenesulfonate and iminodiacetate salts, growth and cation uptake are markedly reduced compared to KCl and NaCl. Calculation of the osmotic potential of salt-treated sections based on measurement of K⁺ and Cl⁻ uptake suggests that the observed increase in tissue osmolality is a result of salt uptake. Similarly, uptake of ions can account for the shift in water potential when sections are incubated in 10 mm KCl. We conclude that the change in growth rate of light-grown, GA₃-treated sections caused by the addition of KCl or NaCl to the incubation medium results solely from decreased water potential of the tissue due to ion uptake.     

Phosphoproteome Analysis of Functional Mitochondria Isolated from Resting Human Muscle Reveals Extensive Phosphorylation of Inner Membrane Protein Complexes and Enzymes

Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins. Combining an efficient mitochondrial isolation protocol with several different phosphopeptide enrichment techniques and LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, including 116 phosphoserine, 23 phosphothreonine, and 16 phosphotyrosine residues. The relatively high number of phosphotyrosine residues suggests an important role for tyrosine phosphorylation in mitochondrial signaling. Many of the mitochondrial phosphoproteins are involved in oxidative phosphorylation, tricarboxylic acid cycle, and lipid metabolism, i.e. processes proposed to be involved in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates for protein kinase A, protein kinase C, casein kinase II, and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria. Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes.Mitochondria are the primary energy-generating systems in eukaryotes. They play a crucial role in oxidative metabolism, including carbohydrate metabolism, fatty acid oxidation, and urea cycle, as well as in calcium signaling and apoptosis (1, 2). Mitochondrial dysfunction is centrally involved in a number of human pathologies, such as type 2 diabetes, Parkinson disease, and cancer (3). The most prevalent form of cellular protein post-translational modifications (PTMs),1 reversible phosphorylation (4–6), is emerging as a central mechanism in the regulation of mitochondrial functions (7, 8). The steadily increasing numbers of reported mitochondrial kinases, phosphatases, and phosphoproteins imply an important role of protein phosphorylation in different mitochondrial processes (9–11).Mass spectrometry (MS)-based proteome analysis is a powerful tool for global profiling of proteins and their PTMs, including protein phosphorylation (12, 13). A variety of proteomics techniques have been developed for specific enrichment of phosphorylated proteins and peptides and for phosphopeptide-specific data acquisition techniques at the MS level (14). Enrichment methods based on affinity chromatography, such as titanium dioxide (TiO₂) (15–17), zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) (18), immobilized metal affinity chromatography (IMAC) (19, 20), and ion exchange chromatography (strong anion exchange and strong cation exchange) (21, 22), have shown high efficiencies for enrichment of phosphopeptides (14). Recently, we demonstrated that calcium phosphate precipitation (CPP) is highly effective for enriching phosphopeptides (23). It is now generally accepted that no single method is comprehensive, but combinations of different enrichment methods produce distinct overlapping phosphopeptide data sets to enhance the overall results in phosphoproteome analysis (24, 25). Phosphopeptide sequencing by mass spectrometry has seen tremendous advances during the last decade (26). For example, MS/MS product ion scanning, multistage activation, and precursor ion scanning are effective methods for identifying serine (Ser), threonine (Thr), and tyrosine (Tyr) phosphorylated peptides (14, 26).A “complete” mammalian mitochondrial proteome was reported by Mootha and co-workers (27) and included 1098 proteins. The mitochondrial phosphoproteome has been characterized in a series of studies, including yeast, mouse and rat liver, porcine heart, and plants (19, 28–31). To date, the largest data set by Deng et al. (30) identified 228 different phosphoproteins and 447 phosphorylation sites in rat liver mitochondria. However, the in vivo phosphoproteome of human mitochondria has not been determined. A comprehensive mitochondrial phosphoproteome is warranted for further elucidation of the largely unknown mechanisms by which protein phosphorylation modulates diverse mitochondrial functions.The percutaneous muscle biopsy technique is an important tool in the diagnosis and management of human muscle disorders and has been widely used to investigate metabolism and various cellular and molecular processes in normal and abnormal human muscle, in particular the molecular mechanism underlying insulin resistance in obesity and type 2 diabetes (32). Skeletal muscle is rich in mitochondria and hence a good source for a comprehensive proteomics and functional analysis of mitochondria (32, 33).The major aim of the present study was to obtain a comprehensive overview of site-specific phosphorylation of mitochondrial proteins in functionally intact mitochondria isolated from human skeletal muscle. Combining an efficient protocol for isolation of skeletal muscle mitochondria with several different state-of-the-art phosphopeptide enrichment methods and high performance LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, many of which have not been reported before. We characterized this mitochondrial phosphoproteome by using bioinformatics tools to classify functional groups and functions, including kinase substrate motifs.