INTRODUCTION

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THERE ARE SEVERAL TYPES of G proteins in the cardiac ventricular myocyte. Each G protein consists of -, -, and -subunits (4). The structures and functions of the -subunits most clearly distinguish the G_s, G_i/o, and G_q/11 forms of the G proteins. G_s is the proximal activator of adenylyl cyclase (AC) and other effector proteins (5). Activation of AC occurs via stimulatory receptor-catalyzed activation of G_s by GTP, resulting in activation of the -subunit and dissociation of the inhibitory G complex. -Adrenergic agonists stimulate AC, increase cAMP concentration, and thereby stimulate cAMP-dependent protein kinase (also called protein kinase A, PKA). The final result is the phosphorylation of effectors such as the L-type Ca²⁺ channel (21). The group of inhibitory G proteins (G_i), comprised of G_i1, G_i2, and G_i3, is distinguished pharmacologically by the ability of pertussis toxin to transfer the ADP-ribose moiety from NAD to the -subunit of the G_i proteins (17). Muscarinic agonists activate G_i, which inhibits AC and therefore decreases Ca²⁺ currents. G_o (G "other") is thought to be very similar to G_i (18) and has been shown to regulate muscarinic receptor affinity for agonists in the brain and heart. G_o is also irreversibly inhibited by pertussis toxin. G_q, a pertussis toxin-insensitive G protein, has been identified in a number of mammalian tissues, including the brain, lung, and heart, and is associated with ₁-adrenergic receptor activation (20).

It has been traditionally believed that muscarinic agonists such as carbachol (CCh) bind to muscarinic receptors to promote formation of an activated G_i-GTP complex. On GTP binding, the heterotrimeric G protein dissociates into two moieties, G_i-GTP and G_i. G_i inhibits AC (10, 15) and modulates intracellular effectors systems such as Ca²⁺ channels. In the heart, this classic view has been challenged by the recent discovery that muscarinic inhibition of Ca²⁺ channels requires the presence of the G_o protein (24). The exact function of G_o in the heart has not been well understood (2). In 1997, Valenzuela et al. (24) studied the muscarinic regulation of Ca²⁺ currents in G_o knockout (G_o/) mouse ventricular myocytes. They found that G_o/ mice have a specific defect in muscarinic regulation of Ca²⁺ current. These results indicate that the G_o protein also plays an important role in the muscarinic regulation of cardiac L-type Ca²⁺ currents. Thus the role of the G_i protein in the muscarinic regulation of the heart has been called into question.

The purpose of the present study is to determine whether G_i is a critical component in the muscarinic regulation of cardiac L-type Ca²⁺ currents. We hypothesize that G_i, like G_o, plays an important role in the muscarinic regulation of Ca²⁺ currents.

	METHODS

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Cell isolation. The generation of G_i2 gene knockout mice has been published (22). The G_i2 protein was not detectable in the G_i2/ mouse heart preparations (23). Age-matched 129Sv wild-type mice and G_i2/ mice were used to isolate cardiac myocytes.

Ca²+ current measurements. Whole cell L-type Ca²⁺ current was measured by using the membrane-ruptured patch configuration (7, 11). To measure Ca²⁺ currents, ventricular cells were placed in a recording chamber (~1.0 ml) on the stage of a Diaphot inverted microscope (Nikon). Ca²⁺ current was recorded using Corning 8161 glass microelectrodes with a tip resistance of 2-4 M when filled with internal solution, which contained (in mM) 96 CsCl, 2 MgCl₂, 10 tetraethylammonium chloride, 14 EGTA, 1 CaCl₂, 20 HEPES, 0.4 Tris-GTP, and 5 Mg-ATP; pH was titrated to 7.1 with CsOH. The bath solution contained (in mM) 135 CsCl, 10 HEPES, 1.8 CaCl₂, 1 MgCl₂, 5 glucose, and 0.01 tetrodotoxin and was titrated to a pH of 7.3 with CsOH. Membrane currents were filtered at 1 kHz with an eight-pole Bessel filter (902LPF, Frequency Devices; Haverhill, MA), converted into digital format using an Axolab 1100 data acquisition system with pCLAMP version 5.5 software (Axon Instruments; Burlingame, CA), and digitally stored for later analysis. The whole cell voltage-clamp protocol used in these studies is similar to that described previously (7). Cell membrane potential was held at 80 mV. After a prepulse to 40 mV for 50 ms, Ca²⁺ currents were elicited by 400-ms depolarizing clamp steps to test potentials ranging from 50 to +50 mV in 10-mV increments. Peak current density was calculated as peak current divided by cell capacitance (in pA/pF). A solenoid-controlled perfusion apparatus was used to change among solutions containing various test agents. A constant flow rate of 1.5 ml/min was used to ensure the exposure of measured cells to the experimental solutions.

Muscarinic receptor radiolabeled ligand binding assay. Individual hearts from wild-type and G_i2/ mice (strain 129Sv; 8-10 wk of age) were excised, rinsed in ice-cold Dulbecco's PBS, minced with scissors, and homogenized in 5 ml of 27% (wt/vol) sucrose, 10 mM Tris · HCl (pH 7.5), and 1 mM EDTA (STE buffer) using a glass Dounce homogenizer (20 strokes with loose and 20 strokes with tight pestle). Homogenates were then passed 10 times through a 26-gauge needle. Particulate matter sedimenting between 5 min at 950 g and 60 min at 100,000 g (cardiac membranes) was collected and resuspended in 0.2 ml of ice-cold STE buffer and diluted in 5 mM MgCl₂, 1 mM EDTA, and 50 mM Tris · HCl (pH 7.5) (binding buffer) to a concentration of 1 mg protein/ml (ca. 10-fold dilution). Duplicate binding reactions were for 60 min at 22°C in 0.5 ml of binding buffer containing 0.1 mg protein and the indicated concentrations of [N-methyl-³H]scopolamine (85 Ci/mmol, Amersham) and, when present, 1 µM ATR to determine nonspecific binding. Binding reactions were stopped and bound N-methyl-scopolamine was separated from unbound by the bovine -globulin/polyethylene glycol coprecipitation method described previously (1, 19). The final membrane pellet was resuspended in 0.4 ml of 0.1 N NaOH, and radioactivity was determined after neutralization with 0.1 ml of 0.4 N HCl in liquid scintillation counter (0.5-ml sample plus 3.0 ml of Parkard Ultima-Flo M scintillation fluid). Under these conditions, binding was proportional to protein concentration and reached equilibrium within 30 min and was stable for up to 90 min.

Statistics. All values were expressed as means ± SE. Student's t-test was used for paired observations, with a P value of <0.05 indicating a statistically significant difference.

	RESULTS

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Basal Ca²+ currents are similar in both wild-type and G_i2 knockout mouse cells. We first examined whether the ventricular myocytes isolated from G_i2/ mice had cell sizes and basal Ca²⁺ current properties comparable to wild-type myocytes. Figure 1A shows Ca²⁺ current tracing recordings from a single ventricular myocyte isolated from a wild-type mouse heart. The cell membrane potential was held at 80 mV and clamped to test potentials from 50 to +50 mV with step increments of 10 mV to elicit the Ca²⁺ current. Figure 1A shows the current tracings from clamping the potential to 40, 0, +20, and +30 mV. Figure 1B displays the current tracings recorded from a G_i2/ mouse myocyte with the same voltage potentials as in Fig. 1A. The configuration of the Ca²⁺ current and the time dependence were very similar in both wild-type and G_i2/ mouse ventricular myocytes. Figure 1C shows the current-voltage relation of the L-type Ca²⁺ currents obtained from these two groups. The data were averaged from 29 wild-type cells (15 mice) and 22 G_i2/ cells (14 mice). The peak current density at a test potential of 0 mV was 7.5 ± 0.2 pA/pF (n = 29) in wild-type mice and 7.6 ± 0.3 pA/pF (n = 22) in G_i2/ mice (P > 0.05). Figure 1C shows that Ca²⁺ currents in wild-type and G_i2/ mouse ventricular myocytes had similar current characteristics, including the maximum current amplitude, peak current potential, and current reversal potential. In addition, the time to the peak current at a test potential of 0 mV was also similar in wild-type (9.2 ± 0.5 ms, n = 29) and G_i2/ mouse ventricular myocytes (8.4 ± 0.5 ms, n = 22, P > 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Comparison of L-type Ca2+ current in wild-type and Gi2/ mouse cardiac ventricular myocytes. A: current tracing recordings from a wild-type mouse ventricular myocyte. Currents were elicited by 350-ms depolarizing clamp steps from a holding potential of 80 mV to test potentials of 40, 0, +20, and +30 mV after a prepulse to 40 mV for 50 ms. B: current tracing recordings from a Gi2/ mouse ventricular myocyte using the same voltage-clamp protocol as in A. Time 0 represents 8 min after cell patch rupture, and the 8-min period was used for the stabilization of L-type Ca2+ current. C: peak Ca2+ current-voltage relation curve from both wild-type and Gi2/ mouse ventricular myocytes. The current amplitude is normalized to cell capacitance (in pA/pF). The data were averaged from 29 wild-type mouse ventricular myocytes (15 mice) and 22 Gi2/ mouse ventricular myocytes (14 mice).

Lack of inhibition of Ca²+ current by a muscarinic cholinergic agonist in G_i2/ mice. To examine the role of the G_i2 protein in the muscarinic regulation of Ca²⁺ current, we first investigated the inhibitory effects of the muscarinic cholinergic agonist CCh on the wild-type mouse ventricular myocyte Ca²⁺ current. Figure 2, A and B, illustrates the effect of CCh (100 µM) on L-type Ca²⁺ current in the wild-type mouse heart. Figure 2A shows representative current tracings recorded under control conditions, perfusion with CCh, and perfusion with CCh plus ATR (10 µM). Figure 2, A and B, demonstrates that CCh inhibits basal L-type Ca²⁺ current in wild-type mouse ventricular myocytes and that this inhibition can be reversed by muscarinic receptor blockade with ATR.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Muscarinic cholinergic suppression of L-type Ca2+ current is absent in Gi2/ ventricular myocytes. Currents were elicited by 350-ms depolarizing clamp steps from a holding potential of 80 mV to a test potential of 0 mV for 350 ms after a prepulse to 40 mV for 50 ms. A: Ca2+ current recording from a wild-type mouse ventricular myocyte under control conditions (control), perfusion with 100 µM carbachol (CCh), and perfusion with 100 µM CCh plus 10 µM atropine (ATR). B: Ca2+ current amplitude time course before and after perfusion with CCh and CCh + ATR. The current amplitude data are derived from the same cell shown in A. Data recording (time = 0) was initiated 8-10 min after a tight seal was formed between the cell membrane and the patch electrode. C: Ca2+ current recording from a Gi2/ mouse ventricular myocyte under control conditions, perfusion with 100 µM CCh, and perfusion with 100 µM CCh + 10 µM ATR. D: Ca2+ current amplitude time course. The current amplitude is derived from the same cell shown in C. Time 0 represents 8 min after cell patch rupture.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Effects of CCh (100 µM) and CCh (100 µM) plus ATR (10 µM) on the L-type Ca2+ currents in both wild-type and Gi2/ mouse ventricular myocytes. Data were averaged from 19 wild-type mouse ventricular myocytes and 8 Gi2/ mouse ventricular myocytes.

-Adrenergic agonist-mediated response is intact in G_i2 gene knockout mouse hearts. We next examined whether the loss of the G_i2 protein interferes with the -adrenergic agonist-mediated stimulation of Ca²⁺ current. We compared the responses of L-type Ca²⁺ currents in ventricular myocytes isolated from either wild-type or G_i2/ mice to the -adrenergic agonist Iso alone and in combination with the cholinergic agonist CCh. Representative examples of wild-type and G_i2/ myocyte current amplitudes in response to Iso and CCh are shown in Fig. 4, A and C. Perfusion with 10 µM Iso increased the L-type Ca²⁺ current in wild-type (Fig. 4A) and G_i2/ (Fig. 4C) mouse ventricular myocytes to a similar extent. This finding indicates that the -adrenergic agonist-mediated response is not affected in G_i gene knockout mice. Perfusion of CCh (100 µM) in the presence of Iso significantly attenuated the Iso-enhanced Ca²⁺ current in wild-type mouse ventricular myocytes (Fig. 4, A and B). However, unlike the wild-type mouse ventricular myocyte, perfusion of the same concentration of CCh in the presence of Iso had no significant effect on Iso-stimulated Ca²⁺ current amplitude in G_i2/ myocytes (Fig. 4, C and D).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   -Adrenergic regulation of L-type Ca2+ current is intact in Gi2/ mouse ventricular myocytes. The voltage-clamp protocol is the same as in Fig. 2. A: Ca2+ current recording from a wild-type mouse ventricular myocyte at control conditions, perfusion with 10 µM isoproterenol (Iso), and perfusion with 10 µM Iso plus 100 µM CCh. B: Ca2+ current amplitude and time course of Ca2+ current recordings of the same cell as in A before and after perfusion of Iso and Iso + CCh. C: Ca2+ current recordings from a Gi2/ mouse ventricular myocyte at control conditions, with perfusion of 10 µM Iso, and perfusion with 10 µM Iso plus 100 µM CCh. D: Ca2+ current amplitude time course. The current amplitude is derived from the same cell shown in C. Time 0 represents 8 min after cell patch rupture.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Effects of Iso and Iso + CCh on L-type Ca2+ currents in both wild-type and Gi2/ mouse ventricular myocytes. Data were averaged from 11 wild-type mouse ventricular myocytes and 12 Gi2/ mouse ventricular myocytes.

G_i2 subunit gene knockout does not alter level of muscarinic receptor expression. To determine the sites and property of muscarinic receptors in G_i2 knockouts, the binding assay of a radiolabeled antagonist ([N-methyl-³H]scopolamine) of the muscarinic receptor to the myocardial membrane protein was performed. Scatchard analysis of the binding of [N-methyl-³H]scopolamine to mouse myocardial membranes showed that the membranes from wild-type mice contained ~84 fmol/mg membrane protein of specific binding sites with an affinity of 0.3 nM and that the membranes from G_i2/ mice contained ~75 fmol/mg membrane protein of specific binding sites with an affinity of 0.2 nM (Fig. 6).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Scatchard analysis of [N-methyl-3H]scopolamine (NMS) binding to wild-type (WT) and Gi2/ mouse myocardial membrane. Membranes (0.1 mg/assay) were incubated with varying concentrations of NMS (85 Ci/mmol, Amersham). Nonspecific binding was determined in presence of 1 µM ATR. Specific binding was the difference between the total binding of NMS in absence of 1 µM ATR and nonspecific binding. Calculation of equilibrium dissociation constants for NMS-receptor interaction was according to equation B/F = Kd1 (B  Bmax), where B is specific bound of ligand, F is free of ligand, Kd is dissociation constant, and Bmax is maximum binding of ligand. Kd of 0.2 and 0.3 nM were calculated for Gi2/ and wild-type cells, respectively. Bmax of 75 fmol/mg was estimated for Gi2/ and 84 fmol/mg for WT cells. Data were averaged from 3 experiments.

	DISCUSSION

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The present study demonstrates that G_i2 is required for the inhibition of L-type Ca²⁺ currents by muscarinic cholinergic agonists in the heart. Whereas the stimulatory G protein pathway activated by Iso was intact, CCh was not able to inhibit either baseline or Iso-stimulated Ca²⁺ currents in the G_i2/ mouse heart. The targeted disruption of G_i2 gene in the G_i2/ mice has been confirmed by ADP ribosylation techniques (23). In the G_i2/ mouse, G_i2 protein was not detectable, but levels of G_o and other G proteins were unchanged (23). Therefore, the observed absence of muscarinic regulation of L-type Ca²⁺ current in the G_i2/ mouse is attributable to the lack of G_i protein and not to the lack of other cell signaling proteins, such as G_o proteins. Although it has been traditionally assumed that G_i is critical for muscarinic regulation of L-type Ca²⁺ current in the heart, we believe that the present study is the first report to confirm this action of G_i using the specific G_i2 knockout mice model.

Regulation of L-type Ca²⁺ current by stimulatory G protein-coupled receptors plays an important role in regulating cardiac function. Activation of the channel by the -adrenergic receptor is a consequence of the activation of AC through G_s. Therefore, cAMP levels rise, PKA is activated, and subsequently the channel is phosphorylated. On the other hand, the mechanism of muscarinic inhibition of the channel is not completely understood (25). In the present study, we found that CCh inhibited the basal (control) Ca²⁺ current amplitude in wild-type mouse ventricular myocytes. This suggests that Ca²⁺ channel current may be regulated in vivo by a combination of sympathetic and parasympathetic mechanisms. Therefore, either the use of a -adrenergic receptor activator such as Iso or a muscarinic cholinoceptor activator such as CCh could affect the Ca²⁺ current amplitude. In the presence of Iso, CCh produced a more profound inhibition of Ca²⁺ current. This phenomenon has been termed as "accentuated antagonism" (13). More experiments are necessary to elucidate the physiological muscarinic regulation of Ca²⁺ currents.

The classic view that muscarinic regulation of Ca²⁺ current requires G_i protein has been challenged by the recent finding that G_o protein also plays an important role in muscarinic regulation of cardiac L-type Ca²⁺ currents (24). The data from the present study, in combination with the findings of Valenzuela et al. (24), suggest that both G_i and G_o may be required for muscarinic cholinergic regulation. However, the apparent "dominant negative" effect of both G protein mutations suggests that G_i and G_o may interact with their respective G protein-coupled signal pathways in a highly complex fashion.

Further investigation as to which second messengers are involved in the muscarinic regulation of L-type Ca²⁺ current will help us to understand the mechanisms of this signaling pathway. It has recently been reported that muscarinic cholinergic regulation of cardiac myocyte Ca²⁺ current is absent in mice with targeted disruption of endothelial nitric oxide (NO) synthase (NOS). Han et al. (13) found that an increase in intracellular cGMP is related to the activity of NOS. This suggests that the Ca²⁺ current is regulated by the NOS and cGMP-dependent protein kinase pathway. In rat ventricular myocytes (3), rabbit sinoatrial nodal cells (14), and atrial ventricular nodal cells (12), muscarinic antagonism of -adrenergic agonist stimulation of Ca²⁺ current was reported to depend on activation of constitutively expressed NOS. On the other hand, Vandecasteele et al. (25) recently found that the NO-cGMP pathway does not contribute significantly to the muscarinic regulation of Ca²⁺ current in human atrial myocytes (25). Although the muscarinic inhibition of AC is impaired in these G_i2/ mice (23), we did not examined whether cAMP or NOS/cGMP is involved in the disruption of muscarinic regulation in the G_i2/ mouse heart in the present study. Recently, we found in another group of cells that addition of intracellular PKA inhibitor only partially reduced the inhibitory effects of CCh (data not shown). This suggests that after acting on G_i protein, CCh may exert its muscarinic regulatory effects through other signal pathways besides the PKA action.

Our study also shows that loss of muscarinic effect on the L-type Ca²⁺ current channel in the G_i2 knockout mouse is neither due to the decreasing of receptor binding sites nor the changing of affinity of receptor to its ligand. The data (Fig. 6) presented here indicate that the numbers of receptor binding sites to [N-methyl-³H]scopolamine in the wild-type and G_i2/ mouse are similar. The affinity of the receptor to its ligand in the G_i2/ mouse myocardial membrane is comparable to that of the wild-type mouse. This suggests that there is no decreasing of the muscarinic receptor binding site in the myocardial membrane of the G_i2 knockout mouse. Loss of the G_i2 subunit by gene knockout does not alter the level of expression of muscarinic receptors in myocardial tissues.

Recently, Jiang et al. (16) also generated G_o gene knockout mice and isolated the ventricular myocytes from these mice. We performed the same experiments as Valenzuela et al. (24). Our preliminary data showed that, similar to their reports, G_o is necessary for muscarinic regulation of Ca²⁺ current in mouse ventricular myocyte, because muscarinic regulation by CCh is absent in G_o gene knockout mouse ventricular myocytes. Our data confirms findings from Valenzuela et al. and also suggests that both G_i and G_o are necessary for muscarinic regulation of Ca²⁺ current in the mouse heart. More recently, it has been reported that G_i2, G_i3, and G_o are all required for normal muscarinic inhibition of the cardiac Ca²⁺ channels in nodal and atrial cultured cardiac cells (28). Further studies are needed to determine how these G proteins are involved in cardiac muscarinic regulation and to clarify the level of interaction of G protein-coupled regulation of L-type Ca²⁺ current in the heart.