María C. García

Modulation of calcium channels of twitch skeletal muscle fibres of the frog by adrenaline and cyclic adenosine monophosphate.

1. Modulation of fast and slow Ca2+ channels of frog skeletal muscle by adrenaline (10(‐6) to 10(‐5) M) and cyclic AMP was investigated using intracellular voltage recordings in intact fibres and a voltage‐clamp technique in cut fibres. 2. In tetraethylammonium (TEA), Cl(‐)‐free Ringer solution, adrenaline increased the maximum rate of rise of Ca2+ spikes by 85% and in a similar solution, peak slow Ca2+ current (ICa,s) by 51%. 3. Application of cyclic AMP to the cut ends of fibres, produced a relative increase of ICa,s of ca. 24%. The effect was maintained for ca. 2 h. 4. Changes in the time course of ICa,s were produced by adrenaline and cyclic AMP: the limiting values of time‐to‐peak current measured as a function of membrane potential were lower (ca. 41% in adrenaline and ca. 34% in cyclic AMP) than those found in control experiments. Also, ICa,s decayed faster in the presence of adrenaline or cyclic AMP. These changes can be explained by exhaustion of Ca2+ in the lumen of transverse tubular system and do not require the assumption of kinetic variations. 5. Fast Ca2+ currents (ICa,f) which could not be blocked by nifedipine were also recorded. Cyclic AMP greatly increased the amplitude of ICa,f but had no obvious effects on ICa,f kinetics. 6. Application of catalytic subunit of cyclic AMP‐dependent protein kinase by diffusion or by pressure injection also increased the amplitude of ICa,s and ICa,f. Pressure injection brought about modifications in the time course of ICa,s that cannot be explained by depletion of Ca2+. 7. Mechanical experiments were performed on single fibres. Nominally Ca2+‐free solutions prevented the development and the maintenance of positive inotropic effect of adrenaline on twitch tension. Development of twitch potentiation was dependent upon the frequency of stimulation. Adrenaline was practically ineffective if no stimulation was applied. 8. It is concluded that both populations of Ca2+ channels are modulated by adrenergic stimulation probably via cyclic AMP, and that twitch potentiation may be mediated by a Ca2+ entry through Ca2+ channels.

Mitochondria regulate inactivation of L‐type Ca2+ channels in rat heart

1 L‐type Ca2+ channels play an important role in vital cell functions such as muscle contraction and hormone secretion. Both a voltage‐dependent and a Ca2+‐dependent process inactivate these channels. Here we present evidence that inhibition of the mitochondrial Ca2+ import mechanism in rat (Sprague‐Dawley) ventricular myocytes by ruthenium red (RR), by Ru360 or by carbonyl cyanide m‐chlorophenylhydrazone (CCCP) decreases the magnitude of electrically evoked transient elevations of cytosolic Ca2+ concentration ([Ca2+]c). These agents were most effective at stimulus rates greater than 1 Hz. 2 RR and CCCP also caused a significant delay in the recovery from inactivation of L‐type Ca2+ currents (ICa). This suggests that sequestration of cytosolic Ca2+, probably near the mouth of L‐type Ca2+ channels, into mitochondria during cardiac contractile cycles, helps to remove the Ca2+‐dependent inactivation of L‐type Ca2+ channels. 3 We conclude that impairment of mitochondrial Ca2+ transport has no impact on either L‐type Ca2+ currents or SR Ca2+ release at low stimulation frequencies (e.g. 0.1 Hz); however, it causes a depression of cytosolic Ca2+ transients attributable to an impaired recovery of L‐type Ca2+ currents from inactivation at high stimulation frequencies (e.g. 3 Hz). The impairment of mitochondrial Ca2+ uptake and subsequent effects on Ca2+ transients at high frequencies at room temperature could be physiologically relevant since the normal heart rate of rat is around 5 Hz at body temperature. The role of mitochondria in clearing Ca2+ in the micro‐domain near L‐type Ca2+ channels could be impaired during high frequencies of heart beats such as in ventricular tachycardia, explaining, at least in part, the reduction of muscle contractility.

Posttranscriptional regulation of the β2-subunit of cardiac L-type Ca2+ channels by MicroRNAs during long-term exposure to isoproterenol in rats.

Introduction and Methods The effects of long-term &bgr;-adrenergic administration on the expression levels of the cardiac L-type Ca2+ channel &bgr;2 subunit, which regulates channel trafficking and function, were characterized in adult rats. Results Systemic administration of isoproterenol (150 mg·kg·h−1) for 2 d led to a 50% increase in the ventricular wet weight-to-body weight ratio (mg/g) and of more than two-fold in the expression of actin protein. In contrast, &bgr;2 subunit protein levels decreased (down to 49%), while mRNA levels remained unchanged. Furthermore, levels of microRNAs (miRs), including miR-21 and miR-132, were upregulated (7.2 and 7.9 fold, respectively). Transfection of these miRs into HEK293 cells attenuated expression of a luciferase reporter gene controlled by a conserved 3′-untranslated region (UTR) of the &bgr;2 subunit (down to 67% and 56%, respectively). Systemic administration of isoproterenol also led to briefer intracellular Ca2+ transients during action potentials measured in isolated cardiomyocytes (down to 65%). Conclusion These results suggest that cardiac L-type Ca2+ channel &bgr;2 subunit protein expression may be downregulated by miRs in response to long-term activation of &bgr;-adrenergic signaling, possibly as an adaptive response in cardiac hypertrophy and sustained &bgr;-adrenergic states.

The β1a subunit regulates the functional properties of adult frog and mouse L-type Ca2+ channels of skeletal muscle

The β1a subunit, one of the auxiliary subunits of CaV1.1 channels, was expressed in COS‐1 cells, purified by electroelution and electrodialysis techniques and identified by Western blot using monoclonal antibodies. The purified β1a subunit strongly interacted in vitro with the alpha interaction domain (AID) of CaV1.1 channels. The actions of the purified β1a subunit on CaV1.1 channel currents were assessed in whole cell voltage clamp experiments performed in vesicles derived from frog and mouse adult skeletal muscle plasma membranes. L‐type inward currents were recorded in solutions containing Ba2+ (IBa). Values of peak IBa were doubled by the β1a subunit in frog and mouse muscle vesicles and the amplitude of the slow component of tail currents was greatly increased. The actions of the β1a subunit on CaV1.1 channel currents reached a steady state within 20 min. The β1a subunit had no effect on the time courses of activation or inactivation of IBa or shifted the current‐voltage relation. Non‐linear capacitive currents were recorded in solutions that contained mostly impermeant ions. Charge movement depended on voltage with average Boltzmann parameters: Qmax+ 28.0 ± 6.6 nC μF−1, V+−58.0 ± 2.0 mV and k+ 15.3± 1.1 mV (n= 24). In the presence of the β1a subunit, these parameters remained unchanged: Qmax+ 29.8 ± 3.5 nC μF−1, V+−54.5 ± 2.2 mV and k+ 16.4± 1.3 mV (n= 21). Overall, the work describes a novel preparation to explore in situ the role of the β1a subunit on the function of adult CaV1.1 channels.

Pharmacological preconditioning by diazoxide downregulates cardiac L-type Ca2+ channels

BACKGROUND AND PURPOSE Pharmacological preconditioning (PPC) with mitochondrial ATP‐sensitive K+ (mitoKATP) channel openers such as diazoxide, leads to cardioprotection against ischaemia. However, effects on Ca2+ homeostasis during PPC, particularly changes in Ca2+ channel activity, are poorly understood. We investigated the effects of PPC on cardiac L‐type Ca2+ channels.

Muscle and motor-skill dysfunction in a K+ channel-deficient mouse are not due to altered muscle excitability or fiber type but depend on the genetic background.

Abstract. The voltage-gated K+ channel Kv3.1 is expressed in skeletal muscle and in GABAergic interneurons in the central nervous system. Hence, the absence of Kv3.1 K+ channels may lead to a phenotype of myogenic or neurogenic origin, or both. Kv3.1-deficient (Kv3.1–/–) 129/Sv mice display altered contractile properties of their skeletal muscles and show poor performance on a rotating rod. In contrast, Kv3.1–/– mice on the (129/Sv×C57BL/6)F1 background display normal muscle properties and perform like wild-type mice. The correlation of poor performance on the rotating rod with altered muscle properties supports the notion that the skeletal muscle dysfunction in Kv3.1–/– 129/Sv mice may be responsible for the impaired motor skills on the rotating rod. Surprisingly, we did not find major differences between wild-type and Kv3.1–/– 129/Sv skeletal muscles in either the resting or action potential, the delayed-rectifier potassium conductance (gK) or the distribution of fast and slow muscle fibers. These findings suggest that the Kv3.1 K+ channel may not play a major role in the intrinsic excitability of skeletal muscle fibers although its absence leads to slower contraction and relaxation and to smaller forces in muscles of 129/Sv Kv3.1–/– mice.

Calciseptine, a Ca2+ Channel Blocker, Has Agonist Actions on L-type Ca2+ Currents of Frog and Mammalian Skeletal Muscle

Abstract. Calciseptine is a natural peptide consisting of 60 amino acids with four disulfide bonds. The peptide is a natural L-type Ca2+-channel blocker in heart and other systems, but its actions in skeletal muscle have not been previously described. The aim of this study is to characterize the effects of calciseptine on L-type Ca2+ channels of skeletal muscle and on contraction. Whole-cell, patch-clamp experiments were performed to record Ca2+ currents (ICa) from mouse myotubes, whereas Vaseline-gap voltage-clamp experiments were carried out to record ICa from frog skeletal muscle fibers. We found that calciseptine acts as a channel agonist in skeletal muscle, increasing peak ICa by 37% and 49% in these two preparations. Likewise, the peptide increased intramembrane charge movement, though it had little effect on contraction. The molecular analysis of the peptide indicated the presence of a local, electrostatic potential that resembles that of the 1,4-dihydropyridine agonist Bay K 8644. These observations suggest that calciseptine shares the properties of 1,4-dihydropyridine derivatives in modulating the permeation of divalent cations through L-type channels.

Regulation of Muscle Cav1.1 Channels by Long-term Depolarization Involves Proteolysis of the α1s Subunit

The effects of long-term depolarization on frog skeletal muscle Cav1.1 channels were assessed. Voltage-clamp and Western-blot experiments revealed that long-term depolarization brings about a drastic reduction in the amplitude of currents flowing through Cav1.1 channels and in the levels of the α1s subunit, the main subunit of muscle L-type channels. The decline of both phenomena was prevented by the action of the protease inhibitors E64 (50 μM) and leupeptin (50 μM). In contrast, long-term depolarization had no effect on β1, the auxiliary subunit of α1s. The levels of mRNAs coding the α1s and the β1 subunits were measured by RNase protection assays. Neither the content of the α1s nor the β1 subunit mRNAs were affected by long-term depolarization, indicating that the synthesis of Cav1.1 channels remained unaffected. Taken together, our experiments suggest that the reduction in the amplitude of membrane currents and in the α1s subunit levels is caused by increased degradation of this subunit by a Ca2+-dependent protease.

Dihydropyridine-sensitive ion currents and charge movement in vesicles derived from frog skeletal muscle plasma membranes.

1 Whole‐cell voltage clamp experiments were performed in vesicles derived from frog skeletal muscle plasma membranes to characterize the electrophysiological properties of dihydropyridine (DHP) receptors. This preparation allows control of the composition of the internal medium and the recording of currents, without the influence of the sarcoplasmic reticulum (SR). 2 In solutions containing Ba2+, Bay K 8644‐sensitive, L‐type inward currents were recorded. Peak Ba2+ currents (IBa) averaged 3.0 μA μF−1 and inactivated in a voltage‐dependent manner. Half‐maximal steady‐state inactivation occurred at −40 mV. No major facilitation of tail currents was observed. 3 The time course of activation of L‐type Ca2+ channels was voltage dependent and 10 times faster than that in muscle fibres; the current density values were also much lower. 4 Lowering [Mg2+]i from 2 to 0.1 mm shifted the time to peak of IBaversus voltage relation by −13 mV. 5 In solutions that contained mostly impermeant ions, non‐linear capacitive currents were recorded. Charge movement with properties resembling charge 1 was observed in polarized vesicles. The charge movement depended on voltage with Boltzmann parameters: Qmax (maximum charge), 45.6 nC μF−1; V(potential at which Q= 0.5Qmax), −58.4 mV; and k (slope factor), 22.3 mV. There was no indication of the presence of Qγ (the ‘hump’ component of charge movement). 6 In depolarized vesicles, non‐linear currents were observed during hyperpolarizing pulses. The currents produced an excessive charge during ‘on’ transients only. Charge during ‘off’ transients was linear from −180 to +60 mV. There was no evidence of the presence of charge 2.

Intracellular Ca2+ transients in delta-sarcoglycan knockout mouse skeletal muscle

BACKGROUNDndelta-Sarcoglycan (delta-SG) knockout (KO) mice develop skeletal muscle histopathological alterations similar to those in humans with limb muscular dystrophy. Membrane fragility and increased Ca(2+) permeability have been linked to muscle degeneration. However, little is known about the mechanisms by which genetic defects lead to disease.nnnMETHODSnIsolated skeletal muscle fibers of wild-type and delta-SG KO mice were used to investigate whether the absence of delta-SG alters the increase in intracellular Ca(2+) during single twitches and tetani or during repeated stimulation. Immunolabeling, electrical field stimulation and Ca(2+) transient recording techniques with fluorescent indicators were used.nnnRESULTSnCa(2+) transients during single twitches and tetani generated by muscle fibers of delta-SG KO mice are similar to those of wild-type mice, but their amplitude is greatly decreased during protracted stimulation in KO compared to wild-type fibers. This impairment is independent of extracellular Ca(2+) and is mimicked in wild-type fibers by blocking store-operated calcium channels with 2-aminoethoxydiphenyl borate (2-APB). Also, immunolabeling indicates the localization of a delta-SG isoform in the sarcoplasmic reticulum of the isolated skeletal muscle fibers of wild-type animals, which may be related to the functional differences between wild-type and KO muscles.nnnCONCLUSIONSndelta-SG has a role in calcium homeostasis in skeletal muscle fibers.nnnGENERAL SIGNIFICANCEnThese results support a possible role of delta-SG on calcium homeostasis. The alterations caused by the absence of delta-SG may be related to the pathogenesis of muscular dystrophy.