RAPID COMMUNICATION
Anchoring protein is required for cAMP-dependent stimulation of L-type Ca2+ channels in rabbit portal vein

Juming Zhong, Joseph R. Hume, and Kathleen D. Keef

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Stimulation of cardiac L-type Ca2+ channels by cAMP-dependent protein kinase (PKA) requires anchoring of PKA to a specific subcellular environment by A-kinase anchoring proteins (AKAP). This study evaluated the possible requirement of AKAP in PKA-dependent regulation of L-type Ca2+ channels in vascular smooth muscle cells using the conventional whole cell patch-clamp technique. Peak Ba2+ current in freshly isolated rabbit portal vein myocytes was significantly increased by superfusion with either 0.5 µM isoproterenol (131 ± 3% of the control value, n = 11) or 10 µM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 114 ± 1%, n = 8). The PKA-induced stimulatory effects of both isoproterenol and 8-BrcAMP were completely abolished by a specific PKA inhibitor KT-5720 (0.2 µM) or by dialyzing cells with Ht 31 (100 µM), a peptide that inhibits the binding of PKA to AKAP. In contrast, Ht 31 did not block the excitatory effect of the catalytic subunit of PKA when dialyzed into the cells. These data suggest that stimulation of Ca2+ channels in vascular myocytes by endogenous PKA requires localization of PKA through binding to AKAP.

whole cell calcium current; vascular smooth muscle; protein kinase A


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CALCIUM INFLUX through L-type Ca2+ channels plays an important role in excitation-contraction coupling of cardiac and vascular smooth muscle cells. In cardiac muscle it is well recognized that L-type Ca2+ channel activity is enhanced by cAMP-dependent protein kinase (PKA). For example, beta -adrenoceptor activation leads to stimulation of adenylyl cyclase via the GTP-binding protein subunit, Gsalpha . The resulting increase in cAMP levels then activates PKA, which stimulates L-type Ca2+ channel activity. Stimulation of channel activity is believed to involve direct phosphorylation of one or more channel subunits, including the alpha 1C subunit of the Ca2+ channel (7, 17).

Recent studies of cardiac L-type Ca2+ channels expressed in HEK cells or in native cardiac cells suggest that PKA-dependent stimulation requires subcellular localization of PKA through association with an A-kinase anchoring protein (AKAP) (7). PKA binds to AKAP via the RII regulatory subunit (10). The PKA binding region of AKAP was first identified in a human thyroid AKAP (Ht-AKAP; see Ref. 2). A 24-amino acid peptide was then synthesized based on the amino acid sequence identified in Ht-AKAP. This peptide, referred to as Ht 31, competes for PKA binding to AKAP (2). Dialysis of cells with Ht 31 has been shown to prevent the potentiation of L-type Ca2+ channels by endogenous PKA in cardiac cells (7) and in skeletal muscle cells (14).

In vascular smooth muscle cells, beta -adrenergic receptor activation also leads to stimulation of L-type Ca2+ channels (13). Furthermore, activation of PKA by other means has been shown to enhance L-type Ca2+ channel activity in vascular myocytes obtained from a variety of different vessels and species (e.g., Refs. 5, 16, 20, 21). However, the requirement of AKAPs for PKA-mediated regulation of L-type Ca2+ channels in vascular smooth muscle cells is still unknown. The present study investigated the role of AKAPs using freshly isolated rabbit portal vein cells and Ht 31 peptides as the probes to disrupt the possible specific subcellular localization of endogenous PKA by AKAPs.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of rabbit portal vein myocytes. Myocytes were isolated using a modification of previously reported methods (20). Male albino rabbits (1.5-2.0 kg) were killed with an intravenous overdose of pentobarbital sodium (50 mg/kg). The portal vein was rapidly removed and cleaned of connective tissue in ice-cold Krebs solution containing (in mM) 125 NaCl, 4.2 KCl, 1.2 MgCl2, 1.8 CaCl2, 11 glucose, 1.2 K2HPO4, 23.8 NaHCO3, and 11 HEPES (pH 7.4 with Trizma base). Cleaned portal vein was then cut into small segments (~4 × 4 mm) and preincubated for 30 min in a shaking water bath at 35°C in a dispersion solution (enzyme-free) containing (in mM) 90 NaCl, 1.2 MgCl2, 1.2 K2HPO4, 20 glucose, 50 taurine, and 5 HEPES (pH 7.1 with NaOH). After preincubation, the segments were incubated in the dispersion solution containing 2 mg/ml collagenase type I (Sigma, St. Louis, MO), 0.5 mg/ml protease type XXVII (Sigma), and 2 mg/ml BSA (Sigma) for 12-15 min at 35°C, and then rinsed four times with the enzyme-free dispersion solution. Smooth muscle cells were dispersed by gentle trituration of the segments with a wide-tipped fire-polished Pasteur pipette. The cell suspension was stored in the enzyme-free dispersion solution containing BSA (1 mg/ml) and Ca2+ (0.1 mM) at 4°C and used within 10 h. The animal use protocol was approved by the Animal Care and Use Committee of the University of Nevada.

Electrophysiology. Ba2+ currents (IBa) in portal vein smooth muscle cells were measured using the conventional whole cell patch clamp. Previous studies from this laboratory have demonstrated that the inward IBa measured from rabbit portal vein myocytes were completely blocked by 10 µM nicardipine, suggesting the presence of predominantly L-type Ca2+ channels in these cells (13). A drop of cell suspension was added to a small recording chamber mounted on the stage of an inverted microscope (Nikon, Japan) and superfused by gravity at a constant rate. All experiments were performed at room temperature (20-22°C). Patch electrodes were made from borosilicate glass pulled with a Sutter P80-PC Flaming/Brown micropipette horizontal puller and fire polished with an MF-83 Narishige microforge. Pipette resistance was 3-5 MOmega when filled with the pipette solution. After establishing the whole cell configuration, cell membrane capacitance and series resistance were determined using a 20-mV hyperpolarizing pulse and were partially compensated. Inward current was elicited by stepping voltage to 0 mV from a holding potential of -70 mV at 30-s intervals using an Axopatch 1D patch-clamp amplifier (Axon Instruments). Voltage-clamp protocols were applied to the cells using the data acquisition package pCLAMP 6 (Axon Instruments) and filtered at 2 kHz (-3dB). Data analysis was performed using the pCLAMP 6 software package.

The bath solution used to record IBa in portal vein cells was composed of (in mM) 117.5 NaCl, 10 tetratethylammonium chloride, 5 BaCl2, 0.5 MgCl2, 5.5 glucose, 5 CsCl, and 10 HEPES (pH 7.4 with NaOH). The pipette solution contained (in mM) 75 glutamic acid, 55 CsCl, 1 K2HPO4, 5 glucose, 5.7 MgSO4, 5 ATP, 0.5 GTP, 10 EGTA, and 10 HEPES (pH 7.2 with CsOH). The osmolality of the solutions (external and internal) was measured and maintained between 290 and 300 mosmol/kgH2O.

Drugs and reagents. Isoproterenol (Iso) and most chemicals were purchased from Sigma. Rp-8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) was obtained from Biolog Life Science Institute (La Jolla, CA). KT-5720, calphostin C, and the catalytic subunit of PKA were obtained from Calbiochem (La Jolla, CA). Ht 31 was synthesized in the Department of Biochemistry, University of Nevada, with the following sequence: Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val-Lys-Ala-Ala-Gly-Ala-Tyr. Ht 31-P was synthesized with the above sequences, except that two isoleucine residues in sequences 10 and 15 were replaced with proline. Drugs insoluble in water were first dissolved in DMSO and were then further diluted in the bathing solution with the final concentration of DMSO <0.2%. DMSO alone at a concentration of 0.2% had no effect on IBa.

Data analysis. Cells were dialyzed with either control pipette solution or pipette solution containing 100 µM Ht 31 or Ht 31-P. The effect of Ht 31 on Ca2+ channel regulation by PKA activation was assessed by comparing channel responses to different drugs in Ht 31 dialyzed cells to matched control cells with the use of identical voltage-clamp protocols. All experimental values are presented as means ± SE (n = number of cells tested). Differences between the values from different groups were compared using both paired and unpaired Student's t-test. Values of P < 0.05 were considered significantly different.


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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES

To determine whether Ht 31 is able to disrupt the PKA-dependent regulation of L-type Ca2+ channels in vascular smooth muscle cells, freshly isolated rabbit portal vein myocytes were dialyzed with normal pipette solution or with pipette solutions containing either Ht 31 (100 µM) or Ht 31-P (100 µM). Ht 31 is a synthetic peptide, which represents residues 493-515 of a human thyroid AKAP and competes for PKA binding to AKAP. Ht 31-P has two isoleucine residues substituted with prolines, which prevent the binding of Ht 31 to PKA (2, 3). IBa was recorded when the cells were stepped to 0 mV from a holding potential of -70 mV. Previous studies from this laboratory indicated that low concentrations of Iso (<1 µM) induce a constant stimulation of Ca2+ channel activity in rabbit portal vein cells (13), possibly through activation of both PKA and protein kinase C (PKC) (23). Thus, in this set of experiments, calphostin C (0.2 µM), a specific PKC inhibitor (8), was always included along with Iso (0.5 µM) unless otherwise stated. Application of calphostin C in the absence of Iso was without effect on IBa (data not shown, n = 5). In control cells, once steady-state current amplitudes were obtained in the whole cell configuration, bath application of Iso led to a significant increase in IBa (mean peak IBa with Iso 131 ± 3% of basal current; Fig. 1). This stimulation could be reversed with the specific PKA inhibitor KT-5720 (Ref. 15, 0.2 µM; data not shown, n = 4). When cells were first dialyzed with Ht 31, Iso was without effect on peak current (Fig. 1). In contrast, when cells were dialyzed with the substituted peptide Ht 31-P, Iso produced the same magnitude of current stimulation as observed in control cells (Fig. 1).


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Fig. 1.   Effect of isoproterenol on Ba2+ current (IBa) in rabbit portal vein myocytes. A: representative recordings from a control cell (a), a cell dialyzed with Ht 31 (100 µM; b), and a cell dialyzed with Ht 31-P (100 µM; c). Currents were elicited by stepping the membrane potential to 0 mV from a holding potential of -70 mV. Current traces in these cells were superimposed before (1) and after (2) superfusion with isoproterenol (Iso; 0.5 µM) in presence of calphostin C (0.1 µM). B: time course of peak current recordings from a control cell and cells dialyzed with different Ht 31 peptides. Iso and calphostin C (cal. c) were added to superfusate when peak current reached a steady state. C: averaged percent changes of peak current in different groups of cells after application of Iso in presence of calphostin C. Numbers in parentheses indicate number of cells tested. Values represent means ± SE. * P < 0.05, significantly different from control.

Because part of the stimulatory effect of Iso on L-type Ca2+ channels may be due to activation of PKC, this component of the Iso response should be independent of PKA binding to AKAP and hence may serve as an effective control to examine the specificity of Ht 31 for PKA. To determine whether Iso could induce a PKA-independent response, Iso was applied in the presence of the PKA blocker KT-5720 (0.2 µM). Addition of Iso (0.5 µM) plus KT-5720 gave rise to a 15 ± 4% increase in peak IBa (n = 7). This stimulatory effect was abolished by calphostin C (data not shown, n = 7), supporting a role for PKC in the actions of Iso. The amount of IBa stimulation observed with Iso plus KT-5720 in cells dialyzed with Ht 31 (18 ± 6% mean peak current increase, n = 12) was not different from the stimulation observed in the absence of Ht 31. These data provide evidence that inhibition of the stimulatory effects of Iso by Ht 31 is specific for the PKA pathway.

To further investigate the role of AKAPs in the actions of PKA on L-type Ca2+ channels, we examined the effects of the PKA activator 8-BrcAMP (10 µM). Bath application of 8-BrcAMP significantly increased peak IBa in control cells (15 ± 3%, n = 9) but had no effect on cells dialyzed with Ht 31 (2 ± 3%, n = 11; Fig. 2). In addition, the increase of peak IBa in control cells by 8-BrcAMP was reversed by KT-5720 (Fig. 2B), indicating that the stimulation of the current by 8-BrcAMP involves activation of PKA.


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Fig. 2.   Effect of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) on IBa in rabbit portal vein myocytes. A: representative recordings from a control cell (a) and a cell dialyzed with Ht 31 (100 µM; b). Currents were elicited by stepping membrane potential to 0 mV from a holding potential of -70 mV. Current traces in these cells were superimposed before (1) and after superfusion with 8-BrcAMP (10 µM; 2) or 8-BrcAMP + KT-5720 (0.2 µM; 3). B: time course of peak current recordings from a control cell and a cell dialyzed with Ht 31. 8-BrcAMP was added to superfusate when peak current reached a steady state. C: averaged percent changes of peak current in both groups of cells after application of 8-BrcAMP. Numbers in parentheses indicate number of cells tested. Values represent means ± SE. * P < 0.05, significantly different from control.

Results from the experiments above suggest that Ht 31 blocks PKA-dependent stimulation of L-type Ca2+ channels in vascular smooth muscle cells. This block is presumed to represent disruption of PKA localization near the Ca2+ channel. Because this action is due to localized levels of PKA, Ht 31 should be ineffective when PKA levels are globally raised by adding exogenous PKA. This assumption was examined by testing the effects of Ht 31 on the actions of purified PKA catalytic subunit introduced into cells by dialysis. In control cells, peak steady-state IBa was reached ~5 min after onset of the whole cell configuration (Fig. 3B). In contrast, when the catalytic subunit of PKA was included in the pipette solution, peak IBa reached a steady-state level ~15 min after onset of the whole cell configuration, and the amplitude of current was significantly larger than that of time-matched control cells (see also Ref. 20). Inclusion of Ht 31 in the pipette solution along with PKA did not reduce the stimulatory effect of exogenous PKA on peak IBa (Fig. 3).


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Fig. 3.   Effect of catalytic subunits of cAMP-dependent protein kinase (PKA) on IBa in rabbit portal vein myocytes. A: representative recordings from a control cell (a), a cell dialyzed with catalytic subunits of PKA (b; 250 U/ml) and a cell dialyzed with catalytic subunits of PKA and Ht 31 (c; 250 U/ml and 100 µM, respectively). Currents were elicited by stepping membrane potential to 0 mV from a holding potential of -70 mV. Current traces in these cells were superimposed at 5 (1) and 20 (2) min after whole cell configuration. B: time course of peak current recordings from a control cell, a cell dialyzed with catalytic subunits of PKA (250 U/ml), and a cell dialyzed with catalytic subunits of PKA (250 U/ml) + Ht 31 (100 µM). C: averaged percent changes of peak current in different groups of cells as a function of time starting from 5 min after whole cell configuration, when peak current in control cells reached a steady state. Values represent means ± SE. * P < 0.05, significantly different from control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subcellular targeting of PKA through association with AKAPs has received extensive attention during recent years. Studies have shown that AKAPs are required for the PKA-dependent modulation of ROMK1 channels in kidney (1), alpha -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate receptors in hippocampal neurons (19), Ca2+-activated K+ channels in airway smooth muscle cells (22), and L-type Ca2+ channels in cardiac cells (7) and skeletal muscle cells (14). A variety of AKAPs have been identified in different cells (2, 4, 6, 9, 11, 12). Localization of PKA is achieved by binding the dimerized regulatory subunits of PKA to a conserved anchoring motif of the AKAPs and targeting the anchored enzyme to a particular environment through the specific targeting domains on each AKAP (10). The catalytic subunits of PKA are released and become active when cAMP molecules bind to the regulatory subunits. Selective compartmentalization of PKA via AKAPs ensures that particular PKA substrates, such as ion channels, can be rapidly and selectively phosphorylated in response to individual stimuli.

Ht 31 is a synthetic peptide representing residues 493-515 of a human thyroid AKAP and encompasses the minimum region required for PKA binding. When Ht 31 binds to the type II regulatory (RII) subunit of PKA it prevents RII binding to AKAP (2). In the present study Ht 31 prevented the stimulation of L-type Ca2+ channels initiated by activation of endogenous PKA with Iso or 8-BrcAMP. Substitution of Ile-502 and Ile-507 in Ht 31 with prolines (referred to as Ht 31-P) abolishes the interaction of Ht 31 with the RII subunits of PKA (2). The ability to neutralize the actions of Ht 31 with this proline substitution has been used as evidence to suggest that the binding of Ht 31 to PKA requires a secondary helical structure (3). Whereas Ht 31 blocked the stimulatory effects observed when endogenous PKA was activated, this same inhibitory peptide was without effect when Ca2+ channels were stimulated through dialysis of cells with the catalytic subunit of PKA. These data suggest that AKAPs permit a level of endogenous PKA activation near the Ca2+ channel that is significantly greater than the overall global level of PKA activation achieved in the cell. On the other hand, when PKA is globally increased through dialysis of the cell with the catalytic subunit of PKA, the need for local targeting of PKA activation is circumvented.

In previous studies we suggested that Iso may activate L-type Ca2+ channels via two pathways, i.e., Gsalpha -mediated activation of PKA and Gbeta gamma -mediated activation of PKC (23). The present study provides additional support for this hypothesis by showing that Iso still stimulates Ca2+ channel current in the presence of PKA blockade and that this effect can be reversed with the PKC blocker calphostin C. When Ht 31 was tested on the two components of the Iso response, we found that Ht 31 blocked the PKA- but not the PKC-mediated actions of Iso. These data provide further evidence for the specificity of the Ht 31 effect. They also suggest that, although PKC localization may require anchoring proteins as well (18), the anchoring motif for PKC binding must differ from that of PKA.

In summary, our results provide additional evidence that PKA activation leads to stimulation of L-type Ca2+ channel current in rabbit portal vein cells. This conclusion is consistent with previous reports by a number of other investigators (5, 13, 16, 20, 21). Our results further indicate that the interaction between endogenous PKA and Ca2+ channels can be disrupted by Ht 31, a peptide that appears to work by preventing the binding of PKA to AKAPs. These data support the hypothesis that anchoring of PKA near the L-type Ca2+ channel is required for PKA-induced stimulation of the channel, although the specific subtypes of AKAPs involved in this process remain unclear.


    ACKNOWLEDGEMENTS

We extend our appreciation to Dr. Kathleen Schegg for the synthesis and purification of Ht 31 and Ht 31-P using the protein/peptide core facility purchased with National Science Foundation Grant BIR-9512482.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-40399 (to K. D. Keef) and HL-49254 (to J. R. Hume). J. Zhong is a recipient of a National Research Service Award postdoctoral fellowship from the NHLBI (HL-10119).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: K. D. Keef, Dept. of Physiology and Cell Biology, Univ. of Nevada, School of Medicine, Reno, NV 89557 (E-mail: kathy{at}physio.unr.edu).

Received 13 May 1999; accepted in final form 15 July 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 277(4):C840-C844
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