Mechanism of voltage-dependent gating in skeletal muscle chloride channels.

Voltage-dependent gating was investigated in a recombinant human skeletal muscle Cl- channel, hCIC-1, heterologously expressed in human embryonic kidney (HEK-293) cells. Gating was found to be mediated by two qualitatively distinct processes. One gating step operates on a microsecond time scale and involves the rapid rearrangement of two identical intramembranous voltage sensors, each consisting of a single titratable residue. The second process occurs on a millisecond time scale and is due to a blocking-unblocking reaction mediated by a cytoplasmic gate that interacts with the ion pore of the channel. These results illustrate a rather simple structural basis for voltage sensing that has evolved in skeletal muscle Cl- channels and provides evidence for the existence of a cytoplasmic gating mechanism in an anion channel analogous to the "ball and chain" mechanism of voltage-gated cation channels.     

G protein-induced trafficking of voltage-dependent calcium channels

Calcium channels are well known targets for inhibition by G protein-coupled receptors, and multiple forms of inhibition have been described. Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane. Imaging experiments in living sensory neurons show that, within seconds of receptor activation, calcium channels are cleared from the membrane and sequestered in clathrin-coated vesicles. Disruption of the L1-CAM-ankyrin B complex with the calcium channel mimics transmitter-induced trafficking of the channels, reduces calcium influx, and decreases exocytosis. Our results suggest that G protein-induced removal of plasma membrane calcium channels is a consequence of disrupting channel-cytoskeleton interactions and might represent a novel mechanism of presynaptic inhibition.     

G proteins and calcium channels in myocardium.

Calcium mobilization has been demonstrated to possess functional importance in myocardial excitation-contraction as well as cellular metabolism. So far, much progress has been made to explore the possibility of involvement of guanine nucleotide binding regulatory protein in the opening and closing of calcium channels as well as intracellular second messenger (cyclic adenosine monophosphate and inositol triphosphate) -mediated calcium mobilization, although such work is still in its preliminary stage, the results have proved highly interesting and significant.     

Calcium-induced and voltage-dependent inactivation of calcium channels in crab muscle fibres

Inactivation of Ca channels was examined in crab muscle fibres using the voltage-clamp method. A satisfactory suppression of outward currents was attempted by the use of K+ blocking agents: TEA, 4AP and Cs ions instead of K+ ions applied extracellularly. The inactivation of Ca current appeared as a bi-exponential process. The faster component had a mean value of the time constant of 50 ms while the second component inactivated at a tenfold slower rate. The extent of inactivation of the faster component increased as the Ca current itself increased in different experimental conditions. Inactivation decreased when ICa was reduced for large applied depolarizations. The time constant of the faster calcium component also depended on the calcium current. Thus the results suggested that Ca2+ entry leads to inactivation of one component of calcium current in crab muscle. Substitution of Ca2+ ions by Sr2+ or Ba2+ ruled out the hypothesis concerning an accumulation process which would explain the decrease of the inward current. The second slower component of Ca current was better described by a voltage-dependent mechanism and its rate was not modified in Ca2+ rich solution or when the inward current was carried by Sr2+ or Ba2+ ions. Thus in crab muscle fibres, inactivation is mediated by both calcium entry and a voltage-gated mechanism.     

Molecular characterization of 1,4-dihydropyridine-sensitive calcium channels of chick heart and skeletal muscle

A monoclonal antibody designated as MAC-L1 immunoprecipitated [3H]PN200-110-labeled calcium channels of chick cardiac and skeletal muscle. On specific immunoprecipitation of 125I-labeled proteins, two large polypeptides (Mr 197,000 and 139,000 for heart, and 172,000 and 135,000 for skeletal muscle, under reducing conditions) were identified as the major components of these channels. Both polypeptides were found to exist together as a complex in 1% digitonin, but to become separated from each other in 1% Triton X-100. The 197 and 172 kDa peptides of cardiac and skeletal muscles, respectively, were photolabeled with [3H]azidopine. Under nonreducing conditions, the 139 kDa polypeptide of heart and the 135 kDa polypeptide of skeletal muscle took on larger molecular weights of 192,000 and 190,000, respectively. The 139 kDa but not the 197 kDa component of the heart was capable of binding to wheat germ agglutinin-Sepharose. Among the polypeptides specifically precipitated by MAC-L1, a 165 kDa peptide of skeletal muscle was phosphorylated by cAMP-dependent protein kinase. In contrast, a minor 99 kDa polypeptide, but not the major 197 kDa polypeptide, of the heart was phosphorylated by this kinase. These results suggest that the dihydropyridine-sensitive cardiac calcium channel has alpha 1 and alpha 2 subunits that are homologous but not identical to those of the skeletal muscle calcium channel.     

Anatomical distribution of voltage-dependent membrane capacitance in frog skeletal muscle fibers

Components of nonlinear capacitance, or charge movement, were localized in the membranes of frog skeletal muscle fibers by studying the effect of 'detubulation' resulting from sudden withdrawal of glycerol from a glycerol-hypertonic solution in which the muscles had been immersed. Linear capacitance was evaluated from the integral of the transient current elicited by imposed voltage clamp steps near the holding potential using bathing solutions that minimized tubular voltage attenuation. The dependence of linear membrane capacitance on fiber diameter in intact fibers was consistent with surface and tubular capacitances and a term attributable to the capacitance of the fiber end. A reduction in this dependence in detubulated fibers suggested that sudden glycerol withdrawal isolated between 75 and 100% of the transverse tubules from the fiber surface. Glycerol withdrawal in two stages did not cause appreciable detubulation. Such glycerol-treated but not detubulated fibers were used as controls. Detubulation reduced delayed (q gamma) charging currents to an extent not explicable simply in terms of tubular conduction delays. Nonlinear membrane capacitance measured at different voltages was expressed normalized to accessible linear fiber membrane capacitance. In control fibers it was strongly voltage dependent. Both the magnitude and steepness of the function were markedly reduced by adding tetracaine, which removed a component in agreement with earlier reports for q gamma charge. In contrast, detubulated fibers had nonlinear capacitances resembling those of q beta charge, and were not affected by adding tetracaine. These findings are discussed in terms of a preferential localization of tetracaine-sensitive (q gamma) charge in transverse tubule membrane, in contrast to a more even distribution of the tetracaine-resistant (q beta) charge in both transverse tubule and surface membranes. These results suggest that q beta and q gamma are due to different molecules and that the movement of q gamma in the transverse tubule membrane is the voltage-sensing step in excitation-contraction coupling.     

Molecular diversity and function of G proteins and calcium channels.

General features of signal transduction by G proteins and structural properties of G-protein-modulated calcium channels are described. Recent results on roles of beta gamma dimers in signal transduction, on the kinetic properties of Gi alpha subunits and structural diversity of Go alpha subunits are discussed, as are the background and current state of our knowledge of the modulation of calcium channels by G proteins.     

Cellular and biochemical features of skeletal muscle in obese Yucatan minipigs

Objective: To examine cellular and biochemical features of skeletal muscle in response to dietary‐induced obesity in a novel Yucatan minipig model of childhood obesity. Research Methods and Procedures: From 4 to 16 months of age, minipigs were fed either a recommended human‐type diet (NF; n = 4) or were overfed a western‐type diet with saturated fat and high‐glycemic index carbohydrates (OF, n = 4). Muscle samples (biceps femoris) were histochemically stained for the identification of intramuscular adipocytes, intramyocellular lipid aggregates (oil red O), and myofiber types (myosin ATPase, succinate dehydrogenase). Gene expressions and/or activities of factors involved in lipogenesis, lipolysis, or energetic metabolism were quantified in muscle. Results: Cross‐sectional areas of myofibers paralleled pig body weight (r = 0.86, p < 0.01). The size of intramuscular adipocytes, the relative proportion of oil red O‐stained fibers, and total muscle lipid content tended (p ≤ 0.10) to increase in response to OF diet. Hormone‐sensitive lipase, carnitine palmityl transferase‐I, and uncoupling protein 2 mRNA levels were lower (p < 0.05) in OF pigs than in NF pigs. Activities of β‐hydroxyacyl‐coenzyme A dehydrogenase and citrate synthase assessing post‐carnitine palmityl transferase I events and the proportion of oxidative myofibers were not altered by OF diet. Activity and gene expression of fatty acid synthase were lower (p < 0.02) in OF pigs than in NF pigs. Discussion: Overfeeding in Yucatan minipigs reduced the expression levels of three catabolic steps in skeletal muscle that are involved also in the etiology of human obesity.     

Identification of putative calcium channels in skeletal muscle microsomes

Saturable binding sites for the labelled calcium antagonist (+/-)[3H]nimodipine were found in guinea-pig hind limb skeletal muscle homogenates. Binding sites were enriched in a microsomal pellet by differential centrifugation of the homogenate. [3H]Nimodipine binding (Kd = 1.5 +/- 0.03 nM, Bmax = 2.1 +/- 0.25 pmol/protein, at 37 degrees C) copurified (6-fold) in this fraction with [3H]ouabain binding (6.6-fold) and 125I-alpha-bungarotoxin binding (5-fold). d-cis-Diltiazem (but not 1-cis-diltiazem) stimulated (+/-) [3H]nimodipine binding (ED50 1 microM) by increasing the Bmax. Binding sites discriminated between the optical enantiomers of 1.4-dihydropyridine calcium antagonists and the optically pure enantiomers of D-600. The data confirm, with biochemical techniques, the presence of 1,4-dihydropyridine and (+/-) D-600 inhibitable calcium channels in skeletal muscle, previously found with electrophysiological techniques.     

The RGK family of GTP-binding proteins: regulators of voltage-dependent calcium channels and cytoskeleton remodeling

RGK proteins constitute a novel subfamily of small Ras-related proteins that function as potent inhibitors of voltage-dependent (VDCC) Ca(2+) channels and regulators of actin cytoskeletal dynamics. Within the larger Ras superfamily, RGK proteins have distinct regulatory and structural characteristics, including nonconservative amino acid substitutions within regions known to participate in nucleotide binding and hydrolysis and a C-terminal extension that contains conserved regulatory sites which control both subcellular localization and function. RGK GTPases interact with the VDCC beta-subunit (Ca(V)beta) and inhibit Rho/Rho kinase signaling to regulate VDCC activity and the cytoskeleton respectively. Binding of both calmodulin and 14-3-3 to RGK proteins, and regulation by phosphorylation controls cellular trafficking and the downstream signaling of RGK proteins, suggesting that a complex interplay between interacting protein factors and trafficking contribute to their regulation.     

Heterogeneity of conductance states in calcium channels of skeletal muscle

The single channel conductance of the dihydropyridine (DHP)-sensitive calcium channel from rabbit skeletal muscle transverse tubules was analyzed in detail using the planar bilayer recording technique. With 0.1 M BaCl2 on both sides of the channel (symmetrical solutions), the most frequent conductance is 12 pS, which is independent of holding potential in the range of -80 to +80 mV. This conductance accounts for approximately 80% of all openings analyzed close to 0 mV. Two additional channels of conductance 9 and 3 pS are also present at all positive potentials, but their relative occurrence close to 0 mV is low. All channels depend on the presence of agonist Bay K 8644 and are inhibited by the antagonist nitrendipine. The relative occurrence of 9 and 3 pS can be increased, and that of 12 pS decreased, by several interventions such as external addition of cholesterol, lectin (wheat germ agglutinin), or calmodulin inhibitor R24571 (calmidazolium). The 9- and 3-pS channels are also conspicuous at positive potentials larger than +40 mV. We suggest that 9- and 3-pS channels are two elementary conductances of the same DHP-sensitive Ca channel. Under most circumstances, these two conductances are gated in a coupled way to generate a channel with a unitary conductance of 12 pS. Interventions tested, including large depolarizations, probably decompose or uncouple the 12-pS channel into 9 and 3 pS.     

Mobility of voltage-dependent ion channels and lectin receptors in the sarcolemma of frog skeletal muscle

The mobility of lectin receptors and of two types of ion channels was studied in skeletal muscles of the frog Rana temporaria. Lectin receptors were labeled with fluorescent derivatives of succinyl-concanavalin A (Con A) or wheat germ agglutinin (WGA), and their mobility was measured by fluorescence recovery after photobleaching. Of the receptors for WGA, approximately 53% were free to diffuse in the plane of the membrane, with an average diffusion coefficient as found in other preparations (D = 6.4 X 10(-11) cm2/s). Con A receptors were not measurably mobile. The mobility of voltage-dependent Na and K (delayed rectifier) channels was investigated with the loose-patch clamp method, coupled with through-the-pipette photodestruction of channels by ultraviolet (UV) light. Na channels were not measurably mobile (D less than or equal to 10(-12) cm2/s). With K channels, photodestruction was followed by a small but consistent recovery of K current, which suggested that some K channels diffused in the plane of the membrane. Our results with K currents are best fit if 25% of the K channels diffuse with D = 5 X 10(-11) cm2/s, with the remainder being immobile. For both Na and K channels, photodestruction by UV was most effective at a wavelength of approximately 289 nm. At this wavelength, the energy density required for an e-fold reduction in the number of functional channels was 0.40 J/cm2 for Na channels and 0.94 J/cm2 for K channels. Irradiation at this wavelength and dose did not measurably diminish the mobility of WGA receptors; hence, the immobility of Na and most K channels is not due to UV irradiation. It is concluded that mobile and immobile membrane proteins coexist in the sarcolemma of frog skeletal muscle, and that voltage-dependent Na and K channels are singled out for immobilization.     

Voltage-dependent calcium channels in skeletal muscle transverse tubules. Measurements of calcium efflux in membrane vesicles

Transverse tubule membranes isolated from rabbit skeletal muscle consist mainly of sealed vesicles that are oriented primarily inside out. These membranes contain a high density of binding sites for 1,4-dihydropyridine calcium channel antagonists. The presence of functional voltage-dependent calcium channels in these membranes has been demonstrated by their ability to mediate 45Ca2+ efflux in response to changes in membrane potential. Fluorescence changes of the voltage-sensitive dye, 3,3'-dipropyl-2,2'-thiadicarbocyanine, have shown that transverse tubule vesicles may generate and maintain membrane potentials in response to establishing potassium gradients across the membrane in the presence of valinomycin. A two-step procedure has been developed to measure voltage-dependent calcium fluxes. Vesicles loaded with 45Ca2+ are first diluted into a buffer designed to generate a membrane potential mimicking the resting state of the cell and to reduce the extravesicular Ca2+ to sub-micromolar levels. 45Ca2+ efflux is then measured upon subsequent depolarization. Flux responses are modulated with appropriate pharmacological specificity by 1,4-dihydropyridines and are inhibited by other calcium channel antagonists such as lanthanum and verapamil.     

Elevations of intracellular calcium reflect normal voltage-dependent behavior,and not constitutive activity,of voltage-dependent calcium channels in gastrointestinal and vascular smooth muscle

In smooth muscle, the gating of dihydropyridine-sensitive Ca²⁺ channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for Ca²⁺ influx and determining the bulk average cytoplasmic Ca²⁺ concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca²⁺ signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (−70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent Ca²⁺ current (I_Ca) and evoked a rise in [Ca²⁺] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca²⁺] increase ([Ca²⁺]_c) was approximately uniform, whereas that of the subplasma membrane space ([Ca²⁺]_PM) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active Ca²⁺ channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca²⁺ channels were not normally constitutively active. The repetitive localized [Ca²⁺]_PM rises (“persistent Ca²⁺ sparklets”) that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca²⁺ channels regulate the bulk average [Ca²⁺]_c. A dihydropyridine blocker of Ca²⁺ channels, nimodipine, which blocked I_Ca and accompanying [Ca²⁺]_c rise, reduced neither the resting bulk average [Ca²⁺]_c (at −70 mV) nor the rise in [Ca²⁺]_c, which accompanied an increased electrochemical driving force on the ion by hyperpolarization (−130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent Ca²⁺ channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca²⁺]_c regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca²⁺].