Channels In Skeletal Muscle Cells Research Articles

Effects of miR-34c-5p on Sodium, Potassium, and Calcium Channel Currents in C2C12 Myotubes.

The aim of this study was to investigate the effects of miR-34c-5p on the main voltage-dependent ion channels in skeletal muscle cells. This study focused on the effects of miR-34c-5p on sodium, potassium, and calcium currents in C2C12 myoblasts. The miR-34c-5p overexpression group, knockdown group, and control group were differentiated for 7days, fused into myotubes, and used for the whole-cell patch clamp recording. Compared with the control group, the whole-cell sodium current density of the other two groups had no significant changes. In the knockdown group, the delayed rectifier potassium current density was increased (statistically significant), and the whole-cell calcium channel current density did not change. In the overexpression group, the change of rectifier potassium current density was not obvious, while the peak calcium channel current density increased (- 9.23 ± 0.95pA/pF, n = 6 cells for the overexpression group vs. - 6.48 ± 0.64pA/pF, n = 7 cells for the control; p < 0.05). Changes in the expression of miR-34c-5p can affect the electrophysiological characteristics of calcium and potassium voltage-gated channels in C2C12 myotubes. Overexpression of miR-34c-5p increased whole-cell L-type calcium channel current (ICa,L), while miR-34c-5p knockdown increased whole-cell delayed rectifier potassium current (IKd).

Spatiotemporal magnetic fields enhance cytosolic Ca2+ levels and induce actin polymerization via activation of voltage-gated sodium channels in skeletal muscle cells

Cellular function is modulated by the electric membrane potential controlling intracellular physiology and signal propagation from a motor neuron to a muscle fiber resulting in muscle contraction. Unlike electric fields, magnetic fields are not attenuated by biological materials and penetrate deep into the tissue. We used complex spatiotemporal magnetic fields (17–70 mT) to control intracellular signaling in skeletal muscle cells. By changing different parameters of the alternating magnetic field (amplitude, inversion time, rotation frequency), we induced transient depolarization of cellular membranes leading to i) Na+ influx through voltage-gated sodium channels (VGSC), ii) cytosolic calcium increase, and iii) VGSC- and ryanodine receptor-dependent increase of actin polymerization. The ion fluxes occurred only, when the field was applied and returned to baseline after the field was turned off. The 30-s-activation-cycle could be repeated without any loss of signal intensity. By contrast, static magnetic fields of the same strength exhibited no effect on myotube Ca2+ levels. Mathematical modeling suggested a role for the alternating magnetic field-induced eddy current, which mediates a local change in the membrane potential triggering the activation of VGSC. These findings might pave the way for the use of complex magnetic fields to improve function of skeletal muscles in myopathies.

Orai and TRP channels in skeletal muscle cells

Modern data concerning expression, localization, biophysical properties, involvement in calcium regulation, and physiological functions of TRP and Orai channels in skeletal muscle cells are analyzed. In skeletal muscles TRPC1/2/3/4/5/6/7, TRPV2/4, TRPM2/7 and Orai1/3 channels are expressed. Activities of TRPC1/3 and TRPV4 facilitate maximal muscle contraction during tetanus. Orai1 channels provide recovery of intracellular calcium stores and are obligatory for proliferation of myoblasts and differentiation of skeletal muscles. TRPC1 knockout results in alterations of the development of skeletal muscles. Enhanced calcium influx via the channels is supposed to be a pathogenic factor of myodystrophy.

Sites of proteolytic processing and noncovalent association of the distal C-terminal domain of CaV1.1 channels in skeletal muscle

In skeletal muscle cells, voltage-dependent potentiation of Ca2+ channel activity requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A-kinase anchoring protein (AKAP15), and the most rapid sites of phosphorylation are located in the C-terminal domain. Surprisingly, the site of interaction of the complex of PKA and AKAP15 with the alpha1-subunit of Ca(V)1.1 channels lies in the distal C terminus, which is cleaved from the remainder of the channel by in vivo proteolytic processing. Here we report that the distal C terminus is noncovalently associated with the remainder of the channel via an interaction with a site in the proximal C-terminal domain when expressed as a separate protein in mammalian nonmuscle cells. Deletion mapping of the C terminus of the alpha1-subunit using the yeast two-hybrid assay revealed that a distal C-terminal peptide containing amino acids 1802-1841 specifically interacts with a region in the proximal C terminus containing amino acid residues 1556-1612. Analysis of the purified alpha1-subunit of Ca(V)1.1 channels from skeletal muscle by saturation sequencing of the intracellular peptides by tandem mass spectrometry identified the site of proteolytic processing as alanine 1664. Our results support the conclusion that a noncovalently associated complex of the alpha1-subunit truncated at A1664 with the proteolytically cleaved distal C-terminal domain, AKAP15, and PKA is the primary physiological form of Ca(V)1.1 channels in skeletal muscle cells.

A novel leucine zipper targets AKAP15 and cyclic AMP-dependent protein kinase to the C terminus of the skeletal muscle Ca2+ channel and modulates its function.

In skeletal muscle, voltage-dependent potentiation of L-type Ca(2+) channel (Ca(V)1.1) activity requires phosphorylation by cyclic AMP-dependent protein kinase (PKA) anchored via an A kinase-anchoring protein (AKAP15). However, the mechanism by which AKAP15 targets PKA to L-type Ca(2+) channels has not been elucidated. Here we report that AKAP15 directly interacts with the C-terminal domain of the alpha(1) subunit of Ca(V)1.1 via a leucine zipper (LZ) motif. Disruption of the LZ interaction effectively inhibits voltage-dependent potentiation of L-type Ca(2+) channels in skeletal muscle cells. Our results reveal a novel mechanism whereby anchoring of PKA to Ca(2+) channels via LZ interactions ensures rapid and efficient phosphorylation of Ca(2+) channels in response to local signals such as cAMP and depolarization.

Calcium regulates L-type Ca2+ channel expression in rat skeletal muscle cells.

Primary skeletal muscle cells were cultured in a normal- (1.8 mM) or high- (4.8 mM) Ca2+ culture medium to determine whether Ca2+ modulates the number of L-type Ca2+ channels. Skeletal myoballs cultured in a normal medium showed, when exposed to a high extracellular [Ca2+], ([Ca2+]e) a transient increase in intracellular [Ca2+] ([Ca2+]i) from a resting concentration of 60 to 160 nM. By day 3, however, when the experiments were made, [Ca2+]i no longer differed from control (pre-exposure to high Ca2+). The maximum charge movements in myoballs incubated in 1.8 and 4.8 mM were 16.4+/-1.05 (n=56) and 24.1+/-1.18 nC/microF (n=58; P<0.01), respectively, and peak Ca2+ currents at 20 mV were -10.8+/-1.09 (n=46) and -12.8+/-0.75 nA/microF (n=82), respectively (P>0.05). The tail current amplitudes in 1.8 and 4.8 mM Ca2+-treated cells were -9.3+/-1.23 and -14.2+/-1.37 nA/microF (P<0.05), respectively, at 10 mV and -15.3+/-1.76 and -23.6+/-2.02 nA/microF (P<0.05), respectively at 60 mV. The maximum binding of [3H]PN200-110 (a radioligand specific for L-type Ca2+ channel alpha1 subunits) in myoballs cultured in 1.8 and 4.8 mM [Ca2+]e was 1.34+/-0.23 and 3.2+/-0.63 pmol/mg protein (n=8; P<0.02), respectively. The increase in [Ca2+]i associated with the increases in charge movements, tail currents and the number of L-type Ca2+ channel alpha1 subunits in skeletal muscle cells cultured in high [Ca2+]e support the concept that extracellular Ca2+ influx modulates the expression of L-type Ca2+ channels in skeletal muscle cells.

Excessive repolarization-dependent calcium currents induced by strong depolarizations in rat skeletal myoballs.

1. Whole-cell patch-clamp recordings were used to study voltage-dependent Ca2+ currents in skeletal myoballs cultured from newborn rats. 2. Depolarizing voltage pulses evoked classical L-type Ca2+ currents, whereas repolarization induced tail currents, whose properties deviated from the expected behaviour of the preceding Ca2+ currents in both voltage dependence and kinetics. 3. Depolarizations of up to +10 mV primarily recruited tail currents that correspond to the Ca2+ channels activated and conducting during the depolarizing pulse, but stronger depolarizations yielded an additional tail current component that exceeded the 'normal' tail current amplitude by several-fold. 4. Activation kinetics of the tail currents were biexponential, with a fast time constant matching the activation time course of the pulse currents (tau approximately 40 ms) and an additional slower component with a voltage-dependent time course that had no kinetic counterpart in the pulse currents (tau approximately 150-600 ms). 5. Both pulse and tail currents were blocked by the dihydropyridine, PN200-110, suggesting that they represent Ca2+ channels of the L-type. 6. We suggest the presence of at least two subsets of dihydropyridine-sensitive Ca2+ channels in skeletal muscle cells. One subset has classical L-type channel characteristics and the other has anomalous gating behaviour that is 'activated' or 'primed' by strong and long-lasting depolarizations without conducting significant Ca2+ current--however, upon repolarization, this subset of channels generates large tail currents.

Voltage-dependent potentiation of L-type Ca2+ channels in skeletal muscle cells requires anchored cAMP-dependent protein kinase.

Skeletal muscle L-type Ca2+ channels respond to trains of brief depolarizations with a strong shift of the voltage dependence of channel activation toward more negative membrane potentials and slowing of channel deactivation. Increased Ca2+ entry resulting from this potentiation of channel activity may increase contractile force in response to tetanic stimuli. This voltage-dependent Ca2+ channel potentiation requires phosphorylation by cAMP-dependent protein kinase (PKA) at a rate that suggests that kinase and channel may be maintained in close proximity through kinase anchoring. A peptide derived from the conserved kinase-binding domain of a PKA-anchoring protein (AKAP) prevents potentiation by endogenous PKA as effectively as inhibition of PKA by a specific peptide inhibitor or by omission of ATP from the intracellular solution. In contrast, a proline-substituted mutant of AKAP peptide has no effect. Potentiation in the presence of 2 microM exogenous catalytic subunit of PKA is unaffected, indicating that kinase anchoring is specifically blocked by the AKAP peptide. No effects of these agents were observed on the level or voltage dependence of basal Ca2+ channel activity before potentiation, suggesting that close physical proximity between the skeletal muscle Ca2+ channel and PKA is critical for voltage-dependent potentiation of Ca2+ channel activity but not for basal activity.