Differential modulation of CaV2.3 Ca2+ channels by Galphaq/11-coupled muscarinic receptors.

CaV2.3 subunits are expressed in neuronal and neuroendocrine cells where they are believed to form native R-type Ca2+ channels. Although R-type currents are involved in triggering neurotransmitter and hormone secretion, little is known about their modulation. Previous studies have shown that muscarinic acetylcholine receptors evoke both inhibition and stimulation of CaV2.3. Muscarinic inhibition of CaV2.3 is mediated by Gbetagamma subunits, whereas stimulation is mediated by pertussis toxin-insensitive Galpha subunits. In the present study, we compared modulation of CaV2.3 by the three Galphaq/11-coupled muscarinic receptors (M1, M3, and M5). Our data indicate that these receptors trigger comparable stimulation of CaV2.3. The signaling pathway that mediates stimulation was meticulously analyzed for M1 receptors. Stimulation is blocked by neutralizing antibodies directed against Galphaq/11, coexpression of the regulatory domain of protein kinase Cdelta (PKCdelta), preactivating PKC with phorbol ester, or pharmacological suppression of PKC with bisindolylmaleimide I. Stimulation of CaV2.3 is Ca(2+)-independent and insensitive to 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö 6976), a specific inhibitor of Ca(2+)-dependent PKC isozymes. These results indicate that muscarinic stimulation of CaV2.3 involves signaling by Galphaq/11, diacylglycerol, and a Ca(2+)-independent PKC. In contrast to stimulation, the magnitude of CaV2.3 inhibition depended on receptor subtype, with M3 and M5 receptors producing much larger CaV2.3 inhibition than M1 receptors. Interestingly, muscarinic inhibition of CaV2.3 was notably enhanced during pharmacological suppression of PKC, suggesting the presence of cross-talk between Gbetagamma-mediated inhibition and PKC-mediated stimulation of R-type channels similar to that described previously for N-type channels.

RGS2 blocks slow muscarinic inhibition of N-type Ca(2+) channels reconstituted in a human cell line.

1. Native N-type Ca(2+) channels undergo sustained inhibition through a slowly activating pathway linked to M1 muscarinic acetylcholine receptors and Galphaq/11 proteins. Little is known concerning the regulation of this slow inhibitory pathway. We have reconstituted slow muscarinic inhibition of N-type channels in HEK293 cells (a human embryonic kidney cell line) by coexpressing cloned alpha1B (Ca(V)2.2) Ca(2+) channel subunits and M1 receptors. Expressed Ca(2+) currents were recorded using standard whole-cell, ruptured-patch techniques. 2. Rapid application of carbachol produced two kinetically distinct components of Ca(2+) channel inhibition. The fast component of inhibition had a time constant of < 1 s, whereas the slow component had a time constant of 5-40 s. Neither component of inhibition was reduced by pertussis toxin (PTX) or staurosporine. 3. The fast component of inhibition was selectively blocked by the Gbetagamma-binding region of beta-adrenergic receptor kinase 1, suggesting that fast inhibition is mediated by Gbetagamma released from Galphaq/11. 4. The slow component of inhibition was selectively blocked by regulator of G protein signalling 2 (RGS2), which preferentially interacts with Galphaq/11 proteins. RGS2 also attenuated channel inhibition produced by intracellular dialysis with non-hydrolysable GTPgammaS. Together these results suggest that RGS2 selectively blocked slow inhibition by functioning as an effector antagonist, rather than as a GTPase-accelerating protein (GAP). 5. These experiments demonstrate that slow muscarinic inhibition of N-type Ca(2+) channels can be reconstituted in non-neuronal cells, and that RGS2 can selectively block slow muscarinic inhibition while leaving fast muscarinic inhibition intact. These results identify RGS2 as a potential physiological regulator of the slow muscarinic pathway.

Muscarinic stimulation of alpha1E Ca channels is selectively blocked by the effector antagonist function of RGS2 and phospholipase C-beta1.

Neuronal alpha1E Ca channel subunits are widely expressed in mammalian brain, where they are thought to form R-type Ca channels. Recent studies have demonstrated that R-type channels contribute to neurosecretion and dendritic Ca influx, but little is known concerning their modulation. Here we show that alpha1E channels are strongly stimulated, and only weakly inhibited, through M1 muscarinic acetylcholine receptors. Both forms of channel modulation are mediated by pertussis toxin-insensitive G-proteins. Channel stimulation is blocked by regulator of G-protein signaling 2 (RGS2) or the C-terminal region of phospholipase C-beta1 (PLCbeta1ct), which have been previously shown to function as GTPase-activating proteins for Galphaq. In contrast, RGS2 and PLCbeta1ct do not block inhibition of alpha1E through M1 receptors. Inhibition is prevented, however, by the C-terminal region of beta-adrenergic receptor kinase 1, which sequesters Gbetagamma dimers. Thus, stimulation of alpha1E is mediated by a pertussis toxin-insensitive Galpha subunit (e.g., Galphaq), whereas inhibition is mediated by Gbetagamma. The ability of RGS2 and PLCbeta1ct to selectively block stimulation indicates these proteins functioned primarily as effector antagonists. In support of this interpretation, RGS2 prevented stimulation of alpha1E with non-hydrolyzable guanosine 5'-0-(3-thiotriphosphate). We also report strong muscarinic stimulation of rbE-II, a variant alpha1E Ca channel that is insensitive to voltage-dependent inhibition. Our results predict that Galphaq-coupled receptors predominantly stimulate native R-type Ca channels. Receptor-mediated enhancement of R-type Ca currents may have important consequences for neurosecretion, dendritic excitability, gene expression, or other neuronal functions.

Biphasic, opposing modulation of cloned neuronal alpha1E Ca channels by distinct signaling pathways coupled to M2 muscarinic acetylcholine receptors.

Neuronal alpha1E subunits are thought to form R-type Ca channels. When expressed in human embryonic kidney cells with M2 muscarinic acetylcholine receptors, Ca channels encoded by rabbit alpha1E exhibit striking biphasic modulation. Receptor activation first produces rapid inhibition of current amplitude and activation rate. However, in the continued presence of agonist, alpha1E currents subsequently increase. Kinetic slowing persists during this secondary stimulation phase. After receptor deactivation, kinetic slowing is quickly relieved, and current amplitude over-recovers before returning toward control levels. These features indicate that inhibition and stimulation of alpha1E are separate processes, with stimulation superimposed on inhibition. Pertussis toxin eliminates inhibition without affecting stimulation, demonstrating that inhibition and stimulation involve distinct signaling pathways. Neither inhibition nor stimulation is altered by coexpression of Ca channel beta2a or beta3 subunits. Stimulation is abolished by staurosporine and reduced by intracellular 5'-adenylylimidodiphosphate, suggesting that phosphorylation is required. However, stimulation does not seem to involve cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, tyrosine kinases, or phosphoinositide 3-kinases. Stimulation does not require a Ca signal, because it is not specifically altered by varying intracellular Ca buffering or by substituting Ba as the charge carrier. In contrast to those formed by alpha1E, Ca channels formed by alpha1A or alpha1B display only inhibition and no stimulation during prolonged activation of M2 receptors. The dual modulation of alpha1E may confer unique physiological properties on native R-type Ca channels. As one possibility, R-type channels may continue to mediate Ca influx during steady inhibition of N-type and P/Q-type channels by muscarinic or other receptors.

Development of multiple calcium channel types in cultured mouse hippocampal neurons.

The development of multiple calcium channel activities was studied in mouse hippocampal neurons in culture, using the patch-clamp technique. A depolarizing pulse (40-50 ms duration) from the holding potential of -80 mV to levels more depolarized than -40 mV produced a low threshold T-type current. The T-type current was observed in 52% of four days in vitro neurons. The number of neurons which expressed T-type current decreased with age of culture, so that the current was detected in only 18% of neurons after 16 days in vitro. The T-type current densities varied between 1.9 pA/pF and 3.29 pA/pF in the mean values during the period studied (4-16 days in vitro). A depolarizing pulse from -80 mV to levels more depolarized than -35 mV evoked a high threshold calcium channel current. The high threshold current density increased in the mean values from 3.9 pA/pF in four days in vitro neurons to 28 pA/pF in 16 days in vitro neurons. We have then examined the effect of nifedipine, omega-Agatoxin IVA and omega-conotoxin GVIA on the high threshold current. Nifedipine (1-5 microM) sensitive current density stayed in the range of 1.9-2.1 pA/pF during 4-16 days in vitro, while omega-Agatoxin IVA (200 nM) sensitive current density increased in the mean values from 1.54 pA/pF in four days in vitro neurons to 21.5 pA/pF in 16 days in vitro neurons. The omega-conotoxin GVIA sensitive N-type channel current was maximum at eight days in vitro (5.44 pA/pF) and it reduced progressively to reach almost half (2.46 pA/pF) in 16 days in vitro neurons. These results showed that diverse subtypes of calcium channels change in density during the early period of culture. We suggest that the temporal expression of each type of channel may be linked to the development of neural activities.

Regulators of G protein signaling attenuate the G protein-mediated inhibition of N-type Ca channels.

Regulators of G protein signaling (RGS) proteins bind to the alpha subunits of certain heterotrimeric G proteins and greatly enhance their rate of GTP hydrolysis, thereby determining the time course of interactions among Galpha, Gbetagamma, and their effectors. Voltage-gated N-type Ca channels mediate neurosecretion, and these Ca channels are powerfully inhibited by G proteins. To determine whether RGS proteins could influence Ca channel function, we recorded the activity of N-type Ca channels coexpressed in human embryonic kidney (HEK293) cells with G protein-coupled muscarinic (m2) receptors and various RGS proteins. Coexpression of full-length RGS3T, RGS3, or RGS8 significantly attenuated the magnitude of receptor-mediated Ca channel inhibition. In control cells expressing alpha1B, alpha2, and beta3 Ca channel subunits and m2 receptors, carbachol (1 microM) inhibited whole-cell currents by approximately 80% compared with only approximately 55% inhibition in cells also expressing exogenous RGS protein. A similar effect was produced by expression of the conserved core domain of RGS8. The attenuation of Ca current inhibition resulted primarily from a shift in the steady state dose-response relationship to higher agonist concentrations, with the EC50 for carbachol inhibition being approximately 18 nM in control cells vs. approximately 150 nM in RGS-expressing cells. The kinetics of Ca channel inhibition were also modified by RGS. Thus, in cells expressing RGS3T, the decay of prepulse facilitation was slower, and recovery of Ca channels from inhibition after agonist removal was faster than in control cells. The effects of RGS proteins on Ca channel modulation can be explained by their ability to act as GTPase-accelerating proteins for some Galpha subunits. These results suggest that RGS proteins may play important roles in shaping the magnitude and kinetics of physiological events, such as neurosecretion, that involve G protein-modulated Ca channels.

The action of diltiazem and gallopamil (D600) on calcium channel current and charge movement in mouse Purkinje neurons.

The dihydropyridines (DHP) receptor forms a high threshold L-type calcium channel in various excitable cells. In skeletal and cardiac muscle cells, a DHP receptor antagonist blocks not only the voltage-gated calcium current but also immobilizes the charge movement linked to the receptor. The DHP receptor is also present in cerebellar Purkinje neurons. Previously, we showed that nifedipine immobilizes a part of the charge movement but has no effect on the calcium channel current recorded in freshly dissociated mice Purkinje neurons. We report here the effect of other families of DHP receptor antagonists, benzothiazepines and phenylalkylamines, on the physiological properties of this receptor in mouse Purkinje neurons.

Effect of SR33805 on barium current and asymmetric intramembrane charge movement in freshly dissociated mouse cerebellar Purkinje neurons.

SR33805 is a novel calcium channel blocker that binds selectively and with high affinity to the alpha 1 subunit of the L-type calcium channel. The binding site for SR33805 is distinct from other classical calcium channel blockers although they interact allosterically. The block by SR33805 of the neuronal L-type calcium current has been reported [Romey, G. and Lazdunski, M., J. Pharmacol. Exp. Ther., 271 (1994) 1348-1352.]. In Purkinje neurons, the L-type calcium current is nearly absent. Nevertheless, we have shown the presence of intramembrane charge movement related to the dihydropyridines (DHP) receptor in these neurons. We show here that SR33805 has no effect on barium currents recorded in Purkinje cells but is a very potent blocker of intramembrane charge movement. It reduces charge movement to 48% of control with an IC50 of 0.5 nM.

Nifedipine-sensitive intramembrane charge movement in Purkinje cells from mouse cerebellum.

1. The intramembrane charge movement was recorded in freshly dissociated Purkinje cells from 14- to 18-day-old mouse cerebellum using the whole-cell voltage clamp technique. 2. After pharmacological elimination of all ionic currents, a depolarizing pulse from a holding potential of -80 mV revealed a transient capacitive outward current at the onset and a transient inward current at the end of the pulse. The amount of charge transferred at the onset (Qon) was equivalent to that moved at the end of the pulse (Qoff). The decay time course of Qon can be fitted by a single exponential curve with a maximum time constant of 1.89 +/- 0.35 ms at 20 mV (n = 11). 3. The charge movement had an S-shaped dependence on test membrane potential, according to a two-state Boltzmann function. The maximum amount (Qmax) of Qon that could be moved was 17.46 +/- 0.83 nC muF-1; the membrane potential at which half the charge movement occurred (V) was 13.48 +/- 2.20 mV and the slope factor (k) was 16.83 +/- 0.84 mV (n = 27). 4. Phenylglyoxal (2 mM), an arginine-specific modifying reagent, reduced Qmax to 60% of control after 20 min treatment. 5. The charge movement was partially immobilized by nifedipine in a dose-dependent manner with an IC50 of 70 nM. The fraction of the nifedipine-sensitive component was 39% of the total charge movement. The potential dependence of the nifedipine-sensitive charge movement could be expressed by a Boltzmann function with values of 7.00 +/- 0.53 nC muF-1 for Qmax, 31.44 +/- 4.23 mV for V and 21.53 +/- 3.18 mV for k (n = 8). 6. The P-type calcium channel specific inhibitor, omega-Aga IVA (250 nM), had no effect on intramembrane charge movement. 7. The above results show that part of the intramembrane charge movement in Purkinje cells may be related to a conformational change of DHP receptors upon membrane depolarization.

Ca2+ entry through acetylcholine receptor channel in dysgenic myotubes.

Skeletal muscles of mutant mice with "muscular dysgenesis" are characterized by excitation-contraction uncoupling resulting from the absence of dihydropyridine receptors. However contraction of the dysgenic myotubes can be evoked by afferent nerve stimulation or by ionophoretic application of acetylcholine (ACh) on the muscle. These contractions are elicited by Ca2+ entry through the ionic channel of the ACh receptor at multiple synaptic contacts. In the present paper, the calcium entry through ACh receptors was compared in cultured normal and dysgenic myotubes. At elevated external calcium concentration (110 mM), the elementary slope conductance of the ACh-activated ionic channel of dysgenic myotubes did not differ from that found in normal myotubes. We conclude that dysgenic muscle contraction induced by nerve stimulation does not result from an abnormal Ca2+ entry across ACh receptors. We discuss the possible involvement of sustained high threshold calcium current (Idys) and of the calcium induced calcium release mechanism in the contractile response related to synaptic activity of dysgenic myotubes.