Dihydropyridine Receptors as Voltage Sensors for a Depolarization-evoked, IP3R-mediated, Slow Calcium Signal in Skeletal Muscle Cells

Abstract

The dihydropyridine receptor (DHPR), normally a voltage-dependent calcium channel, functions in skeletal muscle essentially as a voltage sensor, triggering intracellular calcium release for excitation-contraction coupling. In addition to this fast calcium release, via ryanodine receptor (RYR) channels, depolarization of skeletal myotubes evokes slow calcium waves, unrelated to contraction, that involve the cell nucleus (Jaimovich, E., R. Reyes, J.L. Liberona, and J.A. Powell. 2000. Am. J. Physiol. Cell Physiol. 278:C998–C1010). We tested the hypothesis that DHPR may also be the voltage sensor for these slow calcium signals. In cultures of primary rat myotubes, 10 μM nifedipine (a DHPR inhibitor) completely blocked the slow calcium (fluo-3-fluorescence) transient after 47 mM K+ depolarization and only partially reduced the fast Ca2+ signal. Dysgenic myotubes from the GLT cell line, which do not express the α1 subunit of the DHPR, did not show either type of calcium transient following depolarization. After transfection of the α1 DNA into the GLT cells, K+ depolarization induced slow calcium transients that were similar to those present in normal C2C12 and normal NLT cell lines. Slow calcium transients in transfected cells were blocked by nifedipine as well as by the G protein inhibitor, pertussis toxin, but not by ryanodine, the RYR inhibitor. Since slow Ca2+ transients appear to be mediated by IP3, we measured the increase of IP3 mass after K+ depolarization. The IP3 transient seen in control cells was inhibited by nifedipine and was absent in nontransfected dysgenic cells, but α1-transfected cells recovered the depolarization-induced IP3 transient. In normal myotubes, 10 μM nifedipine, but not ryanodine, inhibited c-jun and c-fos mRNA increase after K+ depolarization. These results suggest a role for DHPR-mediated calcium signals in regulation of early gene expression. A model of excitation-transcription coupling is presented in which both G proteins and IP3 appear as important downstream mediators after sensing of depolarization by DHPR.

Direct Voltage Control of Signaling via P2Y1 and Other Gαq-coupled Receptors

“…The present study demonstrates that the principal voltage sensor lies upstream of phospholipase C or binding of G-protein ␤␥ subunits. Furthermore, there was no evidence for an ability of voltage-gated Ca 2ϩ channels to couple to G-protein cascades, as recently proposed in smooth and skeletal muscle (10,11,62,63). It was interesting that tools commonly used to activate heterotrimeric G-proteins, AlF 4 Ϫ and GTP␥S, induced a voltagedependent Ca 2ϩ response and could initially be taken as evidence that the G-protein itself or its interactions with phospholipase C␤ contribute to the response, as suggested by Ganitkevich and Isenberg (5) in their smooth muscle studies.…”

“…Indeed, a range of physiological voltage waveforms from slow oscillations to action potentials can alter the Ca 2ϩ mobilization induced by P2Y receptor activation (8,9). Constitutive voltage control of phospholipase C and, thus, IP 3 -dependent Ca 2ϩ release has also been described in smooth and skeletal muscle (10,11), inferring that activation of heterotrimeric G-proteins or their receptors can be voltage-dependent in the absence of exogenous agonist. To date, the voltage dependence to GPCR signaling has been most extensively studied in the rat megakaryocyte, where the lack of voltage-gated Ca 2ϩ influx and ryanodine receptors simplifies studies of IP 3 -dependent Ca 2ϩ mobilization (12)(13)(14).…”

Metabotropic Ca 2+ channel-induced Ca 2+ release and ATP-dependent facilitation of arterial myocyte contraction

“…These findings are in excellent agreement with the data obtained in single myocytes, demonstrating that CCICR (the coupling of Ca 2ϩ channels to the SR through the activation of the PLC͞InsP 3 pathway) can be triggered by either membrane depolarization or FPL. The CCICR helps to explain the dependence of InsP 3 -dependent Ca 2ϩ release on the membrane potential observed in smooth muscle (8,9) and other cell types (7,10); however, the possibility that there is a voltage-dependent step at the level of either G protein or PLC activation (8-10) cannot be fully excluded.…”

“…This new Ca 2ϩ -release mechanism depends on the conformational change of L-type Ca 2ϩ channels and the downstream activation of the G protein͞phospholipase C (PLC) cascade, leading to synthesis of InsP 3 and Ca 2ϩ release from the SR (6,7). Although CCICR can be triggered either by direct membrane depolarization in patchclamped myocytes or upon exposure of the cells to a high-K ϩ solution (6), its actual physiological significance is unknown.…”

Altered Gene Expression Pathways in Duchenne Muscular Dystrophy

“…We have previously described that membrane depolarization of skeletal myotubes evokes a fast Ca 2+ transient during the stimuli, that promotes a contractile response through "E-C coupling", and a slow Ca 2+ transient peaking 60-100 seconds later, mostly associated to cell nuclei (Jaimovich et al 2000;Estrada et al 2001;Powell et al 2001;Araya et al 2003;Cardenas et al 2005). Slow Ca 2+ transients are involved in the "E-T coupling" mechanism, which relates membrane depolarization with gene expression (Powell et al 2001;Araya et al 2003;Carrasco et al 2003;Juretic et al 2006;Juretic et al 2007).…”