Gαq Inhibits Cardiac L-type Ca2+ Channels through Phosphatidylinositol 3-Kinase*

Cardiac myocyte contractility is initiated by Ca2+ entry through the voltage-dependent L-type Ca2+ channel (LTCC). To study the effect of Gαq on the cardiac LTCC, we utilized two transgenic mouse lines that selectively express inducible Gαq-estrogen receptor hormone-binding domain fusion proteins (GαqQ209L-hbER or GαqQ209L-AA-hbER) in cardiac myocytes. Both of these proteins inhibit phosphatidylinositol (PI) 3-kinase (PI3K) signaling, but GαqQ209L-AA-hbER cannot activate the canonical Gαq effector phospholipase Cβ (PLCβ). L-type Ca2+ current (ICa,L) density measured by whole-cell patch clamping was reduced by more than 50% in myocytes from both Gαq animals as compared with wild-type cells, suggesting that inhibition of the LTCC by Gαq does not require PLCβ. To investigate the role of PI3K in this inhibitory effect, ICa,L was measured in the presence of various phosphoinositides infused through the patch pipette. Infusion of PI 3,4,5-trisphosphate (PI(3,4,5)P3) into wild-type myocytes did not affect ICa,L, but it fully restored ICa,L density in both Gαq transgenic myocytes to wild-type levels. By contrast, PI 4,5-bisphosphate (PI(4,5)P2) or PI 3,5-bisphosphate had no effect. Infusion with p110β/p85α or p110γ PI3K in the presence of PI(4,5)P2 also restored ICa,L density to wild-type levels. Last, infusion of either PTEN, a PI(3,4,5)P3 phosphatase, or the pleckstrin homology domain of Grp1, which

PI3K or its lipid second messenger PI 3,4,5-trisphosphate (PI(3,4,5)P 3 ) into rat portal vein myocytes increased the I Ca,L amplitude through the LTCC (2,3). Increased LTCC activity in response to activation of PI3K might be due to increased trafficking of the channel to the plasma membrane, as shown using exogenous LTCC proteins expressed in human embryonic kidney 293 cells (4).
Stimulation of G protein-coupled receptors leads to activation of the heterotrimeric G proteins that consist of ␣ and ␤␥ subunits. G␣ and G␤␥ then signal independently to downstream effectors. While the p110␥ and p110␤ PI3Ks are activated by G␤␥ subunits (7,8), the role of G␣ subunits in regulating PI3K is less clear. Recently, we reported that G␣ q coprecipitates with and inhibits the lipid kinase activity of the p110␣/p85␣ PI3K complex (9). Using purified recombinant proteins, we demonstrated that G␣ q binds directly to the enzyme to inhibit its activity. 3 We also found that the G␣ q Q209L mutant, which signals constitutively to its effectors, inhibits p110␣ but not p110␤ (9). Furthermore, G␣ q can inhibit PI3K without activating its canonical effector phospholipase C␤ (PLC␤), as shown by the use of a mutant (G␣ q Q209L-AA) that cannot activate PLC␤ (9 -11).
Using transgenic mice that selectively express an inducible G␣ q Q209L protein in cardiac myocytes, we have demonstrated that activation of G␣ q leads to inhibition of the cardiac I Ca,L . 4 A second line of transgenic mice expressing an inducible G␣ q Q209L-AA protein showed a similar inhibition of the I Ca,L , suggesting a role for PI3K but not PLC␤ in this response. 4 In this study, we used whole-cell patch clamping to further investigate the role of PI3K in mediating G␣ q inhibition of the I Ca,L in myocytes isolated from these transgenic animals. We found that PI(3,4,5)P 3 and some PI3K isoforms can reverse the inhibitory effect of G␣ q on the cardiac LTCC.
G␣ q Q209L-hbER Transgenic Mice-C57BL/6 transgenic mice expressing either G␣ q Q209L-hbER or G␣ q Q209L-AA-hbER in cardiac myocytes under the control of the ␣ myosin heavy chain promoter were described previously. 4 Starting at 8 weeks of age, G␣ q Q209L-hbER animals were injected intraperitoneally with 1 mg of tamoxifen daily for 14 days, and G␣ q Q209L-AA-hbER mice were injected for 28 days. Matching wild-type (WT) littermates that were injected with tamoxifen for an equivalent number of days serve as controls; the WT data shown are pooled from these animals, except where otherwise indicated. All animal-related experimental protocols were approved by the Institutional Animal Care and Use Committee.
Ventricular Myocyte Isolation-Mice were euthanized by intraperitoneal injection of 100 mg/kg sodium pentobarbital, and ventricular myocytes were isolated as previously described. 4 Electrophysiology-Only clearly rod-shaped myocytes were studied. Whole-cell patch clamp recordings used 2-3 M⍀ borosilicate glass pipettes measured prior to sealing (Sutter Instrument), pCLAMP 8 software, the DigiData 1350 interface, and the Axopatch 1D amplifier (Axon Instruments). For the recording of I Ca,L , pipettes contained internal solution (111 mM CsCl, 20 mM tetraethylammonium chloride, 10 mM glucose, 14 mM EGTA, 10 mM HEPES, and 5 mM MgATP, pH 7.2, adjusted with CsOH) and cells were perfused in a Na ϩ -free bath solution (137 mM tetraethylammonium chloride, 1 mM MgCl 2 , 2 mM CaCl 2 , 10 mM HEPES, and 10 mM glucose, pH 7.4, adjusted with tetraethylammonium hydroxide). Except where otherwise noted, the bath solution also contained 1 M 4-HT. The membrane capacity was measured in response to a voltage step from the holding potential, and the current amplitude was divided by cell capacitance to give I Ca,L density in pA/pF. Membrane capacitance is an indirect measure of cell membrane area. This commonly used approach assumes a specific capacitance of 1 F/cm 2 , which could vary slightly depending on physiologic conditions. This variation is expected to be extremely small and well below the detection limit of this technique.
Akt Kinase Assays and Western Blots-Frozen mouse organs were homogenized with a PRO250 (Pro Scientific, Inc., Monroe, CT) in icecold lysis buffer (50 mM HEPES, 1% Triton X-100, 50 mM NaCl, 5 mM EDTA, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovandate, 0.5 mM phenylmethylsulfonyl fluoride, and 10 g/ml each of aprotinin and leupeptin, pH 7.5). Homogenates were centrifuged at 15,000 ϫ g for 30 min at 4°C. Protein concentrations of the supernatants were determined using the Bradford assay (Bio-Rad). Akt activity was assayed in immunoprecipitates starting with 0.5 mg of tissue lysate protein following a method described earlier (12). Western blot signals were visualized and quantitated using the Odyssey Infrared Imaging System with version 1.2 software (LI-COR Biosciences, Lincoln, NE). IRDye800-conjugated second antibodies were from Rockland Immunochemicals (Gilbertsville, PA).
Data Analysis-Values are means Ϯ S.E., and Student's t tests were performed to estimate the significance of the differences between mean values. A value of p Ͻ 0.05 was considered significant. (3,4,5)P 3 on I Ca,L in Cardiac Myocytes-In this study we employed myocytes isolated from two transgenic mouse lines that selectively express silent G␣ q proteins in the heart. These proteins, G␣ q Q209L-hbER and G␣ q Q209L-AA-hbER, are inactive until they bind to 4-HT. Both of them can inhibit PI3K signaling, but the latter cannot activate PLC␤. Activation of these G␣ q proteins in response to injection with tamoxifen, which is converted to 4-HT in animals, causes a large reduction in I Ca,L density in cardiac myocytes from both transgenic animals. 4 This result suggests that inhibition of the cardiac LTCC by G␣ q occurs independently of PLC␤ and may be due to reduced PI3K signaling. This idea is supported by studies showing that PI3Ks stimulate I Ca,L in isolated portal vein myocytes (2, 3). Myocytes were isolated from tamoxifentreated WT, G␣ q Q209L-hbER (QL), and G␣ q Q209L-AA-hbER (QL-AA) mice, and average peak I Ca,L densities were measured by whole-cell patch clamping. The membrane was held at Ϫ50 mV and depolarized for 300 ms to ϩ10 mV. Internal solution with or without phosphoinositides (1 M) was infused through the patch pipette. ** signifies a statistically significant increase in peak I Ca,L density induced by PI(3,4,5)P 3 as compared with the matched control. The number of cells examined in each group is indicated in parentheses.

Effects of PI
As a first test of this hypothesis, we asked if PI(3,4,5)P 3 , the lipid product of PI3K, reverses the depressed I Ca,L in G␣ q Q209L-hbER and G␣ q Q209L-AA-hbER cells. Myocytes were isolated from tamoxifentreated WT and transgenic mice, and internal solution with or without 1 M PI(3,4,5)P 3 was infused into the cells through the patch pipette. The peak I Ca,L density was measured by whole-cell patch clamping at ϩ10 mV following a single depolarizing step of 300 ms duration from a holding potential of Ϫ50 mV. PI(3,4,5)P 3 did not have a significant effect on I Ca,L density in WT myocytes (Fig. 1). However, PI(3,4,5)P 3 significantly increased the I Ca,L density by 2.4-fold in G␣ q Q209L-hbER myocytes and 2.2-fold in the G␣ q Q209L-AA-hbER cells (Fig. 1). The values for PI(3,4,5)P 3 -treated transgenic myocytes were statistically indistinguishable from those observed in WT myocytes treated with or without PI(3,4,5)P 3 (Fig. 1). We performed additional control experiments in G␣ q Q209L-AA-hbER myocytes using other phosphoinositides. Infusion with PI(3,5)P 2 , which is generated from PI 3-phosphate by a PI 5-kinase, had no effect (Fig. 1). Similarly, PI(4,5)P 2 , which is converted to PI(3,4,5)P 3 by PI3K, did not alter the peak I Ca,L density (Fig. 1). These results are consistent with the hypothesis that decreased I Ca,L density in the transgenic myocytes is due to G␣ q -dependent inhibition of PI3K.
We also constructed current density-voltage (I-V) relationships for WT and transgenic myocytes infused with or without PI(3,4,5)P 3 . Activation of I Ca,L was elicited by depolarizing voltage pulses from Ϫ50 mV to ϩ50 mV in 10 mV increments (300 ms duration) from a holding potential of Ϫ50 mV. Fig. 2A shows typical recordings of I Ca,L activation from WT cells in the absence (top panel) or presence (middle panel) of PI(3,4,5)P 3 . The peak I-V curves for both conditions are plotted in the bottom panel ( Fig. 2A). There was no significant difference in I Ca,L density between the two conditions at any of the voltages tested. In contrast, infusion with PI(3,4,5)P 3 resulted in a large enhancement of I Ca,L activation in both the G␣ q Q209L-hbER (Fig. 2B) and G␣ q Q209L-AA-hbER (Fig. 2C) myocytes. The I-V relationships (bottom panels of Fig. 2, B and C) show that I Ca,L density was increased at nearly all of the voltages tested in both groups of myocytes.
To further characterize the action of PI(3,4,5)P 3 in transgenic myocytes, we investigated its time-dependent effect on I Ca,L activation in single cells. In this protocol, the myocyte was infused with or without PI(3,4,5)P 3 and repeatedly depolarized with voltage steps to ϩ10 mV from a holding potential of Ϫ50 mV (300 ms duration). The I Ca,L densities were normalized to the value obtained from the first voltage step FIGURE 2. I-V relationships of myocytes infused with PI(3,4,5)P 3 . Myocytes were isolated from tamoxifen-treated mice. Data were generated using 300-ms depolarizing voltage steps from Ϫ50 to ϩ50 mV in 10-mV increments from a holding potential of Ϫ50 mV. Top and middle panels show representative traces of I Ca,L activation in cells infused with internal solution without (control (Con)) or with 1 M PI(3,4,5)P 3 . Bottom panels plot the mean peak inward current density for each data set at each voltage potential. A, WT, n ϭ 12 for control and n ϭ 7 for PI(3,4,5)P 3 ; B, G␣ q Q209L-hbER (QL), n ϭ 5 for control and n ϭ 7 for PI(3,4,5)P 3 ; C, G␣ q Q209L-AA-hbER (QL-AA), n ϭ 5 for both groups.
following opening of the patch and initiation of whole-cell recording. As shown in the left panel of Fig. 3, the typical "run-down" of I Ca,L density was observed in G␣ q Q209L-hbER myocytes when the patch pipette contained the control internal solution without PI(3,4,5)P 3 . Within 180 s, the normalized I Ca,L density in these cells decreased by about 20%. In contrast, in the presence of PI(3,4,5)P 3 we observed a "run-up" of I Ca,L density in both the G␣ q Q209L-hbER (Fig. 3, middle panel) and G␣ q Q209L-AA-hbER (Fig. 3, right panel) myocytes. I Ca,L density started to decrease after reaching a maximum after ϳ120 s and the rate of decline was similar to that seen in cells infused with the control solution. Interestingly, even the first voltage step elicited a significantly larger I Ca,L in cells infused with PI(3,4,5)P 3 as compared with the control internal solution.
Modulation of I Ca,L by PI3K Isozymes in G␣ q Q209L-AA-hbER Myocytes-We next asked if infusion of purified PI3K proteins into G␣ q Q209L-AA-hbER myocytes has the same effect as PI(3,4,5)P 3 in increasing I Ca,L density. Multiple isoforms of PI3K have been identified in the adult heart of different species (6), and we tested three of them: p110␣/p85␣, p110␤/p85␣, and p110␥. The effects of these PI3K isozymes on the peak I Ca,L density at ϩ10 mV are shown in Fig. 4. In the absence of PI(4,5)P 2 , none of the three PI3K isozymes had an effect on I Ca,L density. However, in the presence of PI(4,5)P 2 , both p110␤/p85␣ and p110␥ induced a significant increase in I Ca,L density (2.6-and 2.2fold, respectively). These increased I Ca,L density values were similar to those observed in WT myocytes (see Fig. 1). Interestingly, infusion with p110␣/p85␣ plus PI(4,5)P 2 had no effect on I Ca,L density (Fig. 4). These results suggest that I Ca,L in the G␣ q transgenic myocytes is modulated by specific PI3K isoforms. Fig. 5 shows typical traces of I Ca,L activation in the presence of PI3K isozymes without (A) or with (B) PI(4,5)P 2 that were used to construct I-V relationships (Fig. 5, C and D). The I-V curves show that infusion of the PI3K isozymes alone did not change I Ca,L density across the entire voltage range examined as compared with the control cells infused with internal solution only (Fig. 5C). In the presence of PI(4,5)P 2 , both p110␤/p85␣ and p110␥ stimulated I Ca,L at nearly all the voltages tested (Fig. 5D). The I-V curve for myocytes infused with p110␣/p85␣ plus PI(4,5)P 2 is nearly identical to the curve obtained from cells infused with PI(4,5)P 2 alone (Fig. 5D).
We also examined the time-dependent effect of PI3K isozymes plus PI(4,5)P 2 on I Ca,L in G␣ q Q209L-AA-hbER myocytes subjected to repeated depolarizing voltage steps. Infusion of p110␣/p85␣ plus PI(4,5)P 2 did not prevent the typical run-down (Fig. 6, left panel). In contrast, a run-up of I Ca,L followed by a slow decrease was observed when either p110␤/p85␣ or p110␥ plus PI(4,5)P 2 were infused into the cells (Fig. 6, middle and right panels).

Reduction of I Ca,L by Decreasing Endogenous PI(3,4,5)P 3 in WT
Myocytes-Since the reduction of I Ca,L density in G␣ q transgenic myocytes can be reversed by infusing exogenous PI(3,4,5)P 3 or some PI3Ks, we hypothesized that depletion of endogenous PI(3,4,5)P 3 in WT myocytes would lower I Ca,L density. Two approaches were used to test this hypothesis. In the first approach, we infused myocytes isolated from WT mice (not treated with tamoxifen) with purified PH-Grp1 protein to sequester intracellular PI(3,4,5)P 3 . PH-Grp1 has been shown to bind specifically to PI(3,4,5)P 3 (13) and has been used to block PI3K signaling (4). In the presence of 20 nM PH-Grp1, the peak I Ca,L density measured after a 300 ms pulse at ϩ10 mV from a holding potential of Ϫ50 mV was 6.6 Ϯ 0.6 pA/pF (n ϭ 7) as compared with 9.6 Ϯ 0.4 pA/pF (n ϭ 7) for cells infused with the control solution. This 31% decrease is statistically significant (t test, p Ͻ 0.01). Shown in Fig. 7A (top two panels) are representative current traces that were used to construct the I-V relationships. The I-V curves show that PH-Grp1 reduced I Ca,L density at nearly all of the voltages tested (Fig. 7A, bottom panel).

. Time-dependent effect of PI(3,4,5)P 3 on I Ca,L activation.
Myocytes were isolated from tamoxifen-treated G␣ q Q209L-hbER (QL) and G␣ q Q209L-AA-hbER (QL-AA) mice. Cells were patched and infused with internal solution without (Con) or with 1 M PI(3,4,5)P 3 . Peak I Ca,L densities were measured following repeated (every 10 s) voltage steps from Ϫ50 mV to ϩ10 mV (300 ms duration). Values shown are average I Ca,L densities normalized to the value of the first I Ca,L recording, which is taken immediately after breaking into the whole-cell mode. Left panel, n ϭ 7; middle and right panels, n ϭ 5 for both groups. Myocytes were isolated from tamoxifen-treated G␣ q Q209L-AA-hbER mice, and average peak I Ca,L densities were measured using the protocol described in the legend of Fig. 1. Cells were infused with internal solution without (Con) or with 20 nM PI3Ks plus or minus 1 M PI(4,5)P 2 . ** signifies a statistically significant increase in I Ca,L density induced by PI3K in the presence of PI(4,5)P 2 as compared with the matched control with enzyme alone. The number of cells analyzed in each group is indicated in parentheses.
In the second approach, we infused myocytes from untreated WT mice with the lipid phosphatase PTEN, which specifically dephosphorylates the D3 position on the inositol ring of PI(3,4,5)P 3 to form PI(4,5)P 2 . When 20 nM purified PTEN protein was infused through the patch pipette, the peak I Ca,L density at ϩ10 mV measured after a 300 ms pulse from a holding potential of Ϫ50 mV was reduced by 28% to 6.5 Ϯ 0.4 pA/pF (n ϭ 20) versus 9.0 Ϯ 0.6 pA/pF (n ϭ 5) for control cells infused with an equivalent volume of PTEN buffer diluted into internal solution. The difference between the two conditions is statistically significant (t test, p Ͻ 0.01). The top two panels in Fig. 7B show sample current traces that were used to construct the I-V relationships for these two conditions (Fig. 7B, bottom panel). The I Ca,L densities were reduced at nearly all of the voltages tested when PTEN was infused into the cells as compared with the control solution. Taken together, these results Mean I-V relationships in the absence (C) or presence (D) of 1 M PI(4,5)P 2 . Con, n ϭ 5; p110␣/p85␣, n ϭ 5; p110␤/p85␣, n ϭ 4; and p110␥, n ϭ 4. FIGURE 6. Time-dependent effect of PI3K isozymes plus PI(4,5)P 2 on I Ca,L activation. Myocytes were isolated from tamoxifen-treated G␣ q Q209L-AA-hbER mice, and I Ca,L densities were measured following repeated depolarization steps and normalized as described in the legend of Fig. 3. Cells were infused with 20 nM PI3K isozymes plus 1 M PI(4,5)P 2 (n ϭ 4 for all three conditions). DECEMBER 2, 2005 • VOLUME 280 • NUMBER 48 demonstrate that reduction of endogenous PI(3,4,5)P 3 levels negatively modulates I Ca,L activation in mouse cardiac myocytes.

G␣ q Inhibition of the L-type Ca 2؉ Channel
Reduced PI3K/Akt Signaling in G␣ q Q209L-AA-hbER Hearts-We previously demonstrated using an Akt activity assay that activation of G␣ q Q209L-AA-hbER in human embryonic kidney 293 cells inhibits PI3K signaling. 4 The protein kinase Akt is a downstream effector of PI3K that is activated by PI(3,4,5)P 3 . Here, we used Akt assays to confirm that PI3K signaling is reduced in hearts of G␣ q Q209L-AA-hbER mice as compared with WT mice. Both groups of animals were injected with tamoxifen for 28 days. Since food intake can affect PI3K signaling in some tissues due to changes in circulating insulin levels, the animals were fasted overnight and then injected with either insulin or saline as a control. Even though cardiac Akt activities were very low in mice injected with saline, we detected a consistent decrease in basal Akt activity in the G␣ q Q209L-AA-hbER hearts as compared with WT (Fig.  8A). G␣ q Q209L-AA-hbER inhibition of PI3K signaling was more obvious in animals treated with insulin. Insulin treatment induced large increases in Akt activity in both WT and G␣ q Q209L-AA-hbER hearts. However, total Akt activity was 33% less in the G␣ q Q209L-AA-hbER hearts as compared with WT (Fig. 8A). The difference in Akt activity is statistically significant (t test, p Ͻ 0.01). In contrast, G␣ q Q209L-AA-hbER did not reduce insulin-induced phosphorylation of Erk1/2 as measured by Western blotting using a phospho-specific antibody (Fig.  8B, bottom panel). Insulin stimulates a modest increase in Erk1/2 phosphorylation in the heart (data not shown). Expression of Akt protein was not reduced in G␣ q Q209L-AA-hbER hearts, as demonstrated by Western blotting of tissue lysates (Fig. 8B, top panel). These results indicate that activation of G␣ q Q209L-AA-hbER results in reduced PI3K/Akt signaling in the heart.

DISCUSSION
Our initial study showing that I Ca,L in G␣ q Q209L-hbER and G␣ q Q209L-AA-hbER transgenic myocytes is similarly depressed as compared with WT myocytes 4 suggested that PLC␤ was not involved in this response. The ability of both of these fusion proteins to inhibit PI3K signaling led us to examine this pathway as a possible mediator of G␣ q inhibition of the LTCC. In this study, we demonstrate that infusion of exogenous PI(3,4,5)P 3 into G␣ q Q209L-hbER and G␣ q Q209L-AA-hbER myocytes completely reverses the inhibition of I Ca,L . I Ca,L is also fully restored in G␣ q Q209L-AA-hbER myocytes infused with certain PI3K isozymes in the presence of their phospholipid substrate. Together, these results support the idea that G␣ q inhibits certain PI3Ks to cause a reduction in I Ca,L . Furthermore, since reduction of endogenous PI(3,4,5)P 3 levels in WT myocytes depresses I Ca,L density, it appears that constitutive PI3K signaling is required for normal LTCC function.
It is well established that activation of G␣ s and subsequent activation of cAMP-dependent protein kinase stimulates cardiac LTCC function. On the other hand, activation of pertussis toxin-insensitive G␣ proteins such as G␣ q/11 inhibits neuronal L-type (14) and N-and P/Q-type (15) Ca 2ϩ channels. G␤␥ subunits released from pertussis toxin-sensitive G proteins also inhibit N-and P/Q-type channels through direct proteinprotein interactions, but they do not bind to or inhibit the LTCC (15). It is not known how activation of G␣ q leads to inhibition of neuronal Ca 2ϩ channels, but our results here using myocytes suggest that it could be due to inhibition of PI3K. Interestingly, a recent study indicates that inhibition of the neuronal LTCC by the M1 muscarinic receptor is mediated by G␣ q/11 but does not appear to involve PLC␤ (16).
Our studies consistently support the concept that G␣ q can inhibit PI3K independently of PLC␤ activation. We initially showed that G␣ q/ 11-coupled ␣ 1A adrenergic receptors inhibit growth factor and insulin activation of PI3K (12,17). Subsequently, we demonstrated that activated G␣ q directly binds to and inhibits the p110␣/p85␣ PI3K 4 (9). By contrast, transfected G␣ q Q209L did not inhibit p110␤ immunoprecipitated from cotransfected cells (9). We have also reported that activated G␣ q does not bind to p110␥, 4 and studies are ongoing in our laboratory to determine whether GTP-bound G␣ q interacts with other isoforms of PI3K. Results in this study show that p110␤/p85␣ or p110␥, but not p110␣/p85␣, reversed G␣ q inhibition of I Ca,L (Figs. 4 and 5). One explanation for this result is that activated G␣ q inhibits only p110␣, and the transgenic myocytes express enough activated G␣ q proteins to neutralize the infused p110␣/p85␣. Activation of PI3K potentiates I Ca,L in rat portal vein myocytes and rat cerebellar granule neurons (1,2). In contrast, we found that exogenous PI(3,4,5)P 3 had no effect on I Ca,L activation in WT mouse cardiac myocytes ( Figs. 1 and 2). We have also found that infusion of canine cardiac myocytes with PI(3,4,5)P 3 does not potentiate I Ca,L activation (data not shown). One possible explanation for these differences between cell types could be that cardiac forms of the LTCC are highly sensitive to the level PI(3,4,5)P 3 and are maximally active at the level found in these cells. Alternatively, PI3K signaling might be maximally activated in cardiac myocytes, so addition of exogenous lipid second messenger would not have an effect. This possibility seems remote because basal Akt activity in the mouse heart is very low but can be strongly increased by insulin treatment (Fig. 8). We have also measured PI3K activity in p110␣, p110␤, or p110␥ immunoprecipitates from freshly prepared heart lysates and found the activities to be very low (data not shown). We believe that LTCC function or localization at the plasma membrane is near maximal in cardiac myocytes, despite the low PI3K activity. Therefore, increasing PI3K signaling does not further stimulate I Ca,L , but inhibition of this signaling pathway by activated G␣ q , PH-Grp1, or PTEN can lead to a reduction in I Ca,L .
Activation of G q -coupled receptors might have complex effects on the modulation of the LTCC. G protein-coupled receptors can activate more than one type of G␣ subunit, and these could modulate LTCC function in diverse ways. Furthermore, some of the released ␤␥ subunits could activate p110␥ or p110␤. In addition, p110␣ can complex with different p85 isoforms, and these heterodimers may be differentially affected by G␣ q . Interestingly, Macrez and co-workers (18) found that angiotensin II stimulation of the portal vein myocyte LTCC is mediated by G␤␥ activation of p110␥. The G␤␥ dimer in question appears to be released from G␣ 13 rather than G␣ q (19). Not all ␤␥ dimers are equivalent in activating PI3K. G␤␥ dimers containing ␤ 5 are least able to activate p110␥ and p110␤, while those containing ␥ 11 are least able to activate p110␥ (8,20). We predict that stimulation of a G q -coupled receptor will negatively modulate the LTCC if (a) the activated G␣ q inhibits p110␣ and (b) its released G␤␥ dimer is a weak activator of p110␤ and p110␥. More studies are needed to determine how cells integrate these competing signals to mount an appropriate Ca 2ϩ channel response.
LTCCs are composed of ␣1, ␤, and ␦/␣2 subunits and, in some forms, an additional ␥ subunit. There are also multiple isoforms of each subunit. The ␣1 subunit forms the pore of the channel and the intracellular ␤ subunit regulates cellular localization. Cardiac LTCCs are mostly composed of ␣1C and ␤2a isoforms, although other ␤ variants are also present in the heart (21). In a heterologous expression system, PI3K signaling stimulated I Ca,L through Akt-mediated phosphorylation of ␤2a subunits, leading to increased trafficking of the LTCC to the plasma membrane (4). Akt specifically phosphorylates the ␤2a subunit on a consensus sequence that is not present in ␤1b, ␤3, or ␤4 (4). Inhibition of Akt by G␣ q may reduce trafficking of the cardiac LTCC to the plasma membrane. Additional studies are planned to determine whether infusion of purified activated Akt proteins reverses the inhibition of I Ca,L in our transgenic myocytes and if these cells show a decreased amount of LTCC proteins in the plasma membrane. Finally, it would be of interest to explore whether G protein-coupled receptors, G␣ q , and p110␣ PI3K form a macromolecular signaling complex with the LTCC at the myocyte plasma membrane so that the current through these channels can be specifically regulated.
In conclusion, results from this study indicate that negative modulation of the LTCC by activated G␣ q in cardiac myocytes is mediated by inhibition of PI3K, perhaps specifically by the p110␣ isoform. Further studies are needed to determine whether L-type and other types of Ca 2ϩ channels present in other excitable cell types are also inhibited by G␣ q through a similar mechanism.