The force of contraction of motor units in skeletal muscle is graded by changing the discharge rate of motor neurons, and cytosolic calcium transients are similarly increased. During single twitches, contraction is not dependent on extracellular calcium, and L-type Ca2+ channels may only function as voltage sensors for initiating Ca2+ release from the sarcoplasmic reticulum. In contrast, forceful tetanic contractions triggered by action potentials at high frequency (20 to 200 Hz) are dependent on extracellular Ca2+ concentration and sensitive to L-type Ca2+ channel antagonists, but the mechanism of regulation of contractile force is unknown. Here we report a large, voltage- and frequency-dependent potentiation of skeletal muscle L-type Ca2+ currents by trains of high-frequency depolarizing prepulses, which is caused by a shift in the voltage-dependence of channel activation to more negative membrane potentials and requires phosphorylation by cyclic AMP-dependent protein kinase in a voltage-dependent manner. This potentiation would substantially increase Ca2+ influx and contractile force in skeletal muscle fibres in response to tetanic stimuli.
Barium currents mediated by the al subunit of the cardiac L-type Ca channel expressed in Chinese hamster ovary (CHO) cells were increased up to 10-fold during dialysis of the cell with the catalytic subunit of cAMP-dependent protein kinase. After partial activation by exogenous kinase, the activity of the al subunit was also reversibly potentiated up to 3.5-fold by prepulses to voltages in the range of 0 to +150 mV. Potentiation at +48 mV developed with a biphasic time course with time constants of 131 ms and 8 s. Reversal at -60 mV was biphasic with half-times of 12 ms and 100 ms and was blocked in the presence of the phosphatase inhibitor okadaic acid. Both the increase in calcium-channel activity during dialysis with kinase and the voltage-dependent potentiation were accompanied by shifts in the voltage dependence of activation to more negative membrane potentials. The increases in Ba current due to protein phosphorylation and to the dihydropyridine Ca channel agonist Bay K8644 were approximately additive. The results show that the al subunit of the cardiac L-type Ca channel is sufficient for substantial modulation of Ca-channel activity by cAMP-dependent protein kinase and for potentiation by state-dependent protein phosphorylation. Voltage-dependent potentiation of the activity of the al subunit may contribute to the increase in contractile force in response to increased rate of stimulation, the positive staircase effect in heart muscle.Calcium ions entering celis through the L-type calcium channels are important regulators of a variety of cellular processes and initiate Ca-induced Ca release and contraction of cardiac muscle (1). Frequency-and voltage-dependent facilitation or potentiation of Ca current by repetitive depolarization has been observed in many cell types including cardiac muscle (2-5). Voltage-dependent facilitation of L-type Ca currents in chromaffin cells activates a previously quiescent Ca channel through phosphorylation by an unknown protein kinase (6). Voltage-and frequency-dependent potentiation of L-type currents in skeletal muscle cells results from rapid, state-dependent phosphorylation by cAMPdependent protein kinase (cA-PK) during positive voltage pulses (7). This potentiation may substantially increase Ca entry during high-frequency trains of action potentials, resulting in increased force during tetanic contractions. Similarly, voltage-dependent potentiation of Ca currents in cardiac cells may contribute to the increase in contractile force in response to increased stimulation rate, the positive staircase effect (2-5), but the mechanism of potentiation of Ca currents by repetitive depolarization is unknown.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Whole cell patch-clamp techniques were used to examine neurokinin receptor modulation of Ca2+ channels in small to medium size dorsal root ganglia neurons (<40 pF) that express mainly N- and L-type Ca2+ currents. Low concentrations of substance P enhanced Ca2+ currents (5-40%, <0.2 microM), while higher concentrations applied cumulatively reversed these enhancements (5-28% reductions, >0.5 microM). This apparent inhibition by high concentrations of substance P was blocked by the administration of the NK3 antagonist SB 235,375 (0.2 microM). The NK1 agonist, [Sar9,Met11]-substance P (0.05 to 1.0 microM) did not alter Ca2+ currents; whereas the NK2 agonist, [betaAla8]-neurokinin A (4-10), enhanced Ca2+ currents (5-36% increase, 0.05-0.5 microM). The enhancement was reversed by the NK2 antagonist MEN 10,376 (0.2 microM) but unaffected by the NK3 antagonist SB 235,375 (0.2 microM). The NK3 agonist [MePhe7]-neurokinin B (0.5-1.0 microM) inhibited Ca2+ currents (6-24% decrease). This inhibition was not prevented by the NK2 antagonist MEN 10,376 (0.2 microM) but was blocked by the NK3 antagonist SB 235,375 (0.2 microM). Both the enhancement and inhibition of Ca2+ currents by neurokinin agonists were reversed by the protein kinase C inhibitor bisindolylmaleimide I HCl (0.2-0.5 microM). Following inhibition of Ca2+ channels by [MePhe7]-neurokinin the facilitatory effect of BayK 8644 (5 microM) was increased and the inhibitory effect of the N-type Ca2+ channel blocker w -conotoxin GVIA (1 microM) was diminished, suggesting that the NK3 agonist inhibits N-type Ca2+ channels. Similarly, block of all but N-type Ca2+ channels, revealed that [betaAla8]-neurokinin A (4-10) enhanced the currents while [MePhe7]-neurokinin B inhibited the currents. Inhibition of all but L-type Ca2+ channels, revealed that [betaAla8]-neurokinin A (4-10) enhanced the currents while [MePhe7]-neurokinin B had no effect. Activation of protein kinase C with low concentrations of phorbol-12,13-dibutyrate enhanced Ca2+ currents, but high concentrations inhibited N- and L-type Ca2+ currents. In summary, these data suggest that in adult rat dorsal root ganglia neurons, NK2 receptors enhance both L- and N-type Ca2+ channels and NK3 receptors inhibit N-type Ca2+ channels and that these effects are mediated by protein kinase C phosphorylation of Ca2+ channels.
Neurokinins released by capsaicin-responsive (C-R) dorsal root ganglia neurons (DRG) may control firing in these neurons by an autofeedback mechanism. Here we used patch clamp techniques to examine the effects of neurokinins on firing properties of dissociated DRG neurons of male rats. In C-R neurons that generated only a few action potentials (APs, termed phasic) in response to long depolarizing current pulses (600 ms), substance P (SP, 0.5 μM) lowered the AP threshold by 11.0 ±0.3 mV and increased firing from 1.1±0.7 APs to 5.2±0.6 APs. In C-R tonic neurons that fire multiple APs, SP elicited smaller changes in AP threshold (6.0±0.1 mV reduction) and the number of APs (11±1 vs. 9±1 in control). The effects of SP were similar to the effect of heteropodatoxin II (0.05 μM) or low concentrations of 4-aminopyridine (50 μM) that block A-type K + currents. A selective NK 2 agonist, [βAla 8 ]-neurokinin A (4-10) (0.5 μM), mimicked the effects of SP. The effects of SP in C-R phasic neurons were fully reversed by an NK 2 receptor antagonist (MEN10376, 0.5 μM) but only partially by a protein kinase C (PKC) inhibitor (bisindolylmaleimide, 0.5 μM). An NK 3 selective agonist ([MePhe 7 ]-neurokinin B, 0.5 μM), an NK 1 selective agonist ([Sar 9 , Met 11 ]-substance P, 0.5 μM) or activation of PKC with phorbol 12, 13-dibutyrate (0.5 μM) did not change firing. Our data suggest that the excitability of C-R phasic afferent neurons is increased by activation of NK 2 receptors and intracellular signaling mediated only in part by PKC.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.