Rem inhibits skeletal muscle EC coupling by reducing the number of functional L-type Ca2+ channels.

In skeletal muscle, the L-type voltage-gated Ca(2+) channel (1,4-dihydropyridine receptor) serves as the voltage sensor for excitation-contraction (EC) coupling. In this study, we examined the effects of Rem, a member of the RGK (Rem, Rem2, Rad, Gem/Kir) family of Ras-related monomeric GTP-binding proteins, on the function of the skeletal muscle L-type Ca(2+) channel. EC coupling was found to be weakened in myotubes expressing Rem tagged with enhanced yellow fluorescent protein (YFP-Rem), as assayed by electrically evoked contractions and myoplasmic Ca(2+) transients. This impaired EC coupling was not a consequence of altered function of the type 1 ryanodine receptor, or of reduced Ca(2+) stores, since the application of 4-chloro-m-cresol, a direct type 1 ryanodine receptor activator, elicited myoplasmic Ca(2+) release in YFP-Rem-expressing myotubes that was not distinguishable from that in control myotubes. However, YFP-Rem reduced the magnitude of L-type Ca(2+) current by approximately 75% and produced a concomitant reduction in membrane-bound charge movements. Thus, our results indicate that Rem negatively regulates skeletal muscle EC coupling by reducing the number of functional L-type Ca(2+) channels in the plasma membrane.

G-protein inhibition of N- and P/Q-type calcium channels: distinctive elementary mechanisms and their functional impact.

Voltage-dependent G-protein inhibition of presynaptic Ca(2+) channels is a key mechanism for regulating synaptic efficacy. G-protein betagamma subunits produce such inhibition by binding to and shifting channel opening patterns from high to low open probability regimes, known respectively as "willing" and "reluctant" modes of gating. Recent macroscopic electrophysiological data hint that only N-type, but not P/Q-type channels can open in the reluctant mode, a distinction that could enrich the dimensions of synaptic modulation arising from channel inhibition. Here, using high-resolution single-channel recording of recombinant channels, we directly distinguished this core contrast in the prevalence of reluctant openings. Single, inhibited N-type channels manifested relatively infrequent openings of submillisecond duration (reluctant openings), which differed sharply from the high-frequency, millisecond gating events characteristic of uninhibited channels. By contrast, inhibited P/Q-type channels were electrically silent at the single-channel level. The functional impact of the differing inhibitory mechanisms was revealed in macroscopic Ca(2+) currents evoked with neuronal action potential waveforms (APWs). Fitting with a change in the manner of opening, inhibition of such N-type currents produced both decreased current amplitude and temporally advanced waveform, effects that would not only reduce synaptic efficacy, but also influence the timing of synaptic transmission. On the other hand, inhibition of P/Q-type currents evoked by APWs showed diminished amplitude without shape alteration, as expected from a simple reduction in the number of functional channels. Variable expression of N- and P/Q-type channels at spatially distinct synapses therefore offers the potential for custom regulation of both synaptic efficacy and synchrony, by G-protein inhibition.

Ca(2+) channel modulation by recombinant auxiliary beta subunits expressed in young adult heart cells.

L-type Ca(2+) channels contribute importantly to the normal excitation-contraction coupling of physiological hearts, and to the functional derangement seen in heart failure. Although Ca(2+) channel auxiliary beta(1-4) subunits are among the strongest modulators of channel properties, little is known about their role in regulating channel behavior in actual heart cells. Current understanding draws almost exclusively from heterologous expression of recombinant subunits in model systems, which may differ from cardiocytes. To study beta-subunit effects in the cardiac setting, we here used an adenoviral-component gene-delivery strategy to express recombinant beta subunits in young adult ventricular myocytes cultured from 4- to 6-week-old rats. The main results were the following. (1) A component system of replication-deficient adenovirus, poly-L-lysine, and expression plasmids encoding beta subunits could be optimized to transfect young adult myocytes with 1% to 10% efficiency. (2) A reporter gene strategy based on green fluorescent protein (GFP) could be used to identify successfully transfected cells. Because fusion of GFP to beta subunits altered intrinsic beta-subunit properties, we favored the use of a bicistronic expression plasmid encoding both GFP and a beta subunit. (3) Despite the heteromultimeric composition of L-type channels (composed of alpha(1C), beta, and alpha(2)delta), expression of recombinant beta subunits alone enhanced Ca(2+) channel current density up to 3- to 4-fold, which argues that beta subunits are "rate limiting" for expression of current in heart. (4) Overexpression of the putative "cardiac" beta(2a) subunit more than halved the rate of voltage-dependent inactivation at +10 mV. This result demonstrates that beta subunits can tune inactivation in the myocardium and suggests that other beta subunits may be functionally dominant in the heart. Overall, this study points to the possible therapeutic potential of beta subunits to ameliorate contractile dysfunction and excitability in heart failure.

Differential occurrence of reluctant openings in G-protein-inhibited N- and P/Q-type calcium channels.

Voltage-dependent inhibition of N- and P/Q-type calcium channels by G proteins is crucial for presynaptic inhibition of neurotransmitter release, and may contribute importantly to short-term synaptic plasticity. Such calcium-channel modulation could thereby impact significantly the neuro-computational repertoire of neural networks. The differential modulation of N and P/Q channels could even further enrich their impact upon synaptic tuning. Here, we performed in-depth comparison of the G-protein inhibition of recombinant N and P/Q channels, expressed in HEK 293 cells with the m2 muscarinic receptor. While both channel types display classic features of G-protein modulation (kinetic slowing of activation, prepulse facilitation, and voltage dependence of inhibition), we confirmed previously reported quantitative differences, with N channels displaying stronger inhibition and greater relief of inhibition by prepulses. A more fundamental, qualitative difference in the modulation of these two channels was revealed by a modified tail-activation paradigm, as well as by a novel "slope" analysis method comparing time courses of slow activation and prepulse facilitation. The stark contrast in modulatory behavior can be understood within the context of the "willing-reluctant" model, in which binding of G-protein betagamma subunits to channels induces a reluctant mode of gating, where stronger depolarization is required for opening. Our experiments suggest that only N channels could be opened in the reluctant mode, at voltages normally spanned by neuronal action potentials. By contrast, P/Q channels appear to remain closed, especially over these physiological voltages. Further, the differential occurrence of reluctant openings is not explained by differences in the rate of G-protein unbinding from the two channels. These two scenarios predict very different effects of G-protein inhibition on the waveform of Ca(2+) entry during action potentials, with potentially important consequences for the timing and efficacy of synaptic transmission.

Signaling mechanisms underlying muscarinic receptor-mediated increase in contraction rate in cultured heart cells.

We have investigated the mechanisms by which stimulation of cardiac muscarinic receptors result in paradoxical stimulatory effects on cardiac function, using cultured neonatal rat ventricular myocytes as a model system. Application of low concentrations of carbachol (CCh) (EC50 = 35 nM) produced an atropine-sensitive decrease in spontaneous contraction rate, while, in cells pretreated with pertussis toxin, higher concentrations of CCh (EC50 = 26 microM) elicited an atropine-sensitive increase in contraction rate. Oxotremorine, an m2 muscarinic acetylcholine receptor (mAChR) agonist, mimicked the negative but not the positive chronotropic response to CCh. Reverse transcription followed by polymerase chain reaction carried out on mRNA obtained from single cells indicated that ventricular myocytes express mRNA for the m1, m2, and, possibly, m4 mAChRs. The presence of m1 and m2 mAChR protein on the surface membranes of the cultured ventricular myocytes was confirmed by immunofluorescence. The CCh-induced positive chronotropic response was significantly inhibited by fluorescein-tagged antisense oligonucleotides directed against the m1, but not the m2 and m4, mAChR subtypes. The response was also inhibited by antisense oligonucleotides against Gqalpha protein. Finally, inhibition of CCh-induced phosphoinositide hydrolysis with 500 microM neomycin or 5 microM U73122 completely abolished the CCh-induced positive chronotropic response. These results are consistent with the stimulatory effects of mAChR activation on the rate of contractions in cultured ventricular myocytes being mediated through the m1 mAChR coupled through Gq to phospholipase C-induced phosphoinositide hydrolysis.

Does mammalian heart contain only the M2 muscarinic receptor subtype?

Five muscarinic acetylcholine receptor (mAChR) subtypes, m1-m5, have been cloned and sequenced to date. The question as to which mAChR subtypes exist in mammalian heart has been studied extensively and is still under considerable debate. We used the reverse transcriptase-polymerase chain reaction to amplify mRNA from adult rat ventricular myocytes, and found that these cells express mRNA for m1 and m2 mAChRs. Immunocytochemical analysis confirmed that m1 and m2, but not m3, mAChR proteins are present on the surface of these cells. Finally, the functional significance of these receptors was examined. Administration of the m1 mAChR antagonist pirenzepine inhibited the stimulatory effect of the muscarinic agonist carbachol on Ca transients. These findings are consistent with the presence of at least two mAChR subtypes in mammalian heart, m1 and m2, and suggest that activation of m1 mAChRs is involved in the stimulatory effects of muscarinic agonists in mammalian heart.

Molecular and functional identification of m1 muscarinic acetylcholine receptors in rat ventricular myocytes.

The expression of muscarinic acetylcholine receptor (mAChR) subtypes in freshly isolated adult rat ventricular myocytes was investigated by reverse transcription of cellular mRNA followed by amplification of cDNA using the polymerase chain reaction (PCR). After reverse-transcriptase PCR, bands were obtained corresponding to the expected sizes for the m1 and m2 but not for the m3 to m5 mAChRs. The identity of the m1 and m2 bands was confirmed by single-cell PCR, restriction digest mapping, and Southern blot analysis. The presence of m1 and m2, but not m3, mAChR protein in these cells was shown by indirect immunofluorescence studies using subtype-specific antibodies. It was further investigated whether the identified m1 mAChR was responsible for the stimulatory effects on Ca2+ transients by high concentrations of carbachol ( > 10 mumol/L) known to occur in these cells. In pertussis toxin-treated ventricular myocytes electrically stimulated at 1 Hz, carbachol (300 mumol/L) increased the basal Ca2+ level from 96 +/- 7 to 118 +/- 8 nmol/L and the peak Ca2+ transient level from 519 +/- 32 to 640 +/- 36 nmol/L (mean +/- SEM P < .05 for both, n = 8). These effects of carbachol on Ca2+ transients were antagonized by 10 nmol/L pirenzepine, an m1 mAChR-selective antagonist. In contrast, the m2 mAChR-selective antagonist methoctramine (up to 100 nmol/L) did not inhibit the response. These results are the first to use single-cell PCR to probe cardiomyocyte-specific gene expression and indicate that m1 mAChRs are expressed on adult rat ventricular myocytes in addition to m2 mAChRs. The results further suggest that m1 mAChRs mediate the stimulatory responses on Ca2+ transients to high concentrations of cholinergic agonists seen in these cells.