Published online before print March 20, 2008, doi: 10.1161/CIRCRESAHA.108.171736
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© 2008 American Heart Association, Inc.
UltraRapid Communication |
Role of CaVβ Subunits, and Lack of Functional Reserve, in Protein Kinase A Modulation of Cardiac CaV1.2 Channels
From the Department of Physiology and Cellular Biophysics (H.M.C.), Columbia University, College of Physicians and Surgeons, New York; and Department of Biomedical Engineering (J.M., T.N., D.T.Y., H.M.C.), Johns Hopkins University, Baltimore, Md.
Correspondence to Henry M. Colecraft, Columbia University, College of Physicians and Surgeons, Department of Physiology and Cellular Biophysics, 630 W 168th St, P&S 7-422, New York, NY 10032. E-mail hc2405{at}columbia.edu
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Abstract |
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Protein kinase A (PKA)-mediated enhancement of L-type calcium currents (ICa,L) is essential for sympathetic regulation of the heartbeat and is the classic example of channel regulation by phosphorylation, and its loss is a common hallmark of heart failure. Mechanistic understanding of how distinct CaV channel subunits contribute to PKA modulation of ICa,L has been intensely pursued yet remains elusive. Moreover, critical features of this regulation such as its functional reserve (the surplus capacity available for modulation) in the heart are unknown. Here, we use an overexpression paradigm in heart cells to simultaneously identify the impact of auxiliary CaVβs on PKA modulation of ICa,L and to gauge the functional reserve of this regulation in the heart. CaV1.2 channels containing wild-type β2a or a phosphorylation-deficient mutant (β2a,AAA) were equally upregulated by PKA, discounting a necessary role for β phosphorylation. Nevertheless, channels reconstituted with β2a displayed a significantly diminished PKA response compared with other β isoforms, an effect explainable by a uniquely higher basal Po of β2a channels. Overexpression of all βs increased basal current density, accompanied by a concomitant decrease in the magnitude of PKA regulation. Scatter plots of fold increase in current against basal current density revealed an inverse relationship that was conserved across species and conformed to a model in which a large fraction of channels remained unmodified after PKA activation. These results redefine the role of β subunits in PKA modulation of CaV1.2 channels and uncover a new design principle of this phenomenon in the heart, vis à vis a limited functional reserve.
Key Words: Ca channel • β subunit • PKA • modulation
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Introduction |
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L-type (CaV1.2) channels are important determinants of cardiac excitability and indispensable for excitation–contraction coupling in the heart.1 Fitting with their central role in cardiac biology, tuning CaV1.2 channel activity is a prominent method for physiological and therapeutic regulation of the heartbeat.2 A classic example of physiological modulation is the upregulation of cardiac CaV1.2 channel activity in response to β-adrenergic receptor stimulation, an effect mediated through activation of protein kinase A (PKA).2–4 This phenomenon is an important contributor to sympathetic regulation of the heartbeat, and its loss is a harmful hallmark of heart failure.2,5 PKA-mediated regulation of cardiac CaV1.2 channels represents the original example of channel modulation by phosphorylation, and mechanistic understanding of this phenomenon has been widely pursued for more than 3 decades.3,4,6
Despite years of intense study, many key features of how activated PKA upregulates CaV1.2 channel activity remain unknown. Native CaV1.2 channels are comprised minimally of a pore-forming 
1C protein assembled in a 1:1 ratio with auxiliary CaVβ and 2
subunits.2,7 CaVβ subunits are necessary for trafficking CaV1 to the plasma membrane and also greatly influence channel gating properties.8,9 There are 4 distinct CaVβ isoforms (β1 to β4), each with multiple splice variants.10–15 Structurally, CaVβs have a conserved core containing SH3 and guanylate kinase–like motifs,16–18 appended by nonconserved N and C termini. CaVβ2 subunits are predominant in the heart,8,12,19,20 although other isoforms are present as well.21
It has been hypothesized that the CaVβ component of CaV1.2 channels may critically regulate the extent of PKA modulation of ICa,L.22 This has been postulated as a possible reason for why PKA upregulation of ICa,L is stronger in the heart compared with other tissue, such as smooth muscle or neurons. However, this hypothesis has not been rigorously tested. CaVβs could influence PKA modulation of ICa,L in several ways. First, direct phosphorylation of CaVβ could be important. Importantly, CaVβ2a is PKA phosphorylated both in vivo and in vitro on 3 serine residues located on the unique C terminus (Ser459, Ser478, Ser479).23 It has been suggested that phosphorylation of CaVβ2a on Ser478 and Ser479 is necessary for PKA modulation of ICa,L but only in a channel configuration in which the pore-forming 1C lacks a C terminus.22 This idea has not been confirmed, and its relevance to regulation of the endogenous cardiac CaV1.2 channel is uncertain. Moreover, the β2a PKA phosphorylation sites are not conserved in other types of β subunits, raising the pertinent question of whether L-type channels reconstituted with these other isoforms differ substantively in the extent of PKA regulation. To date, this concept has not been tested. A second way in which CaVβs could differentially influence PKA modulation stems from the observation that distinct CaVβs can confer markedly different basal single-channel open probability (Po) to CaV1.2 channels in the heart.8 Because PKA increases ICa,L by elevating single-channel Po,24 β subunits that produce an already high basal Po might be expected to show a diminished PKA response owing to a reduced dynamic range of modulation. Finally, it is possible that diverse CaVβs could promote subtly distinct structural configurations of 1C that, nevertheless, produce substantial differences in CaV1.2 channel response to PKA. In short, the full scope of how CaVβ’s might influence PKA-dependent modulation of CaV1.2 channels is unknown.
Efforts to understand how PKA phosphorylation of CaV1.2 channel subunits lead to increased ICa,L have been hampered by a singular challenge: it has not been possible to reconstitute robust, reproducible functional PKA modulation of recombinant ICa,L in heterologous systems.25–28 By contrast, biochemical phosphorylation of CaV1.2 channels on 1C (Ser1928) and β2a subunits is readily demonstrated in heterologous systems.29,30 The disconnect between biochemical and functional modulation of recombinant channels is puzzling and suggests that CaV1.2 channel phosphorylation may not be sufficient for increasing ICa,L. Here, we circumvent ambiguities associated with recapitulating PKA modulation of ICa,L in heterologous cells by overexpressing CaVβs in isolated heart cells, where the channel regulation is most robust and reproducible. Serendipitously, this experimental paradigm yields additional dividends beyond just identifying the impact of CaVβs on PKA upregulation of ICa,L. The functional reserve, defined as the excess capacity available for PKA modulation of CaV1.2 channels in the heart, is unknown. Yet, this information could provide important new perspectives to 2 disparate phenomena: (1) the decrement in PKA modulation of ICa,L observed in the heart failure and (2) the difficulty in reconstituting PKA regulation of recombinant CaV1.2 channels in heterologous cells. Overexpressing CaVβs in the heart cells increases the number of functional channels in the sarcolemma,8 providing an opportunity to probe the functional reserve of PKA-mediated enhancement of ICa,L.
We find that the CaVβ subunit composition of cardiac CaV1.2 channels can powerfully impact the extent of PKA modulation of ICa,L but only in an indirect manner: channels reconstituted with β2a exhibit a significantly diminished PKA-mediated upregulation compared with other β isoforms because their elevated basal Po results in a lower dynamic range for channel tuning. Furthermore, we found that cells with overexpressed CaVβs displayed a diminished PKA-mediated increase in ICa,L that could be explained by assuming the appearance of an increased fraction of nonresponsive channels, suggesting a very limited functional reserve for channel regulation.
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Materials and Methods |
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Generation of Plasmid and Adenoviral Constructs
Construction of adenoviral vectors encoding β1b–green fluorescent protein (GFP), β2a-GFP, β3-GFP, and β4-GFP has been previously described.8 To create Ad-β2a,AAA-GFP, we used overlap extension PCR to introduce point mutations (S459A, S478A, S479A) into human β2a. The mutated β2a subunit was cloned into the adenoviral shuttle plasmid vector, pAdCMV EGFP-N3, using NheI and BamHI sites, generating pAdCMV β2a,AAA-GFP. The adenoviral vector Ad-β2a,AAA-GFP was subsequently generated using Cre-lox recombination as previously described.8,31
Cell Culture and Gene Transfection
Primary cultures of adult guinea pig and Sprague–Dawley rat heart cells were isolated by enzymatic digestion using a Langendorff perfusion apparatus. Animal treatment and use were in accordance with the guidelines of the Johns Hopkins University Animal Care and Use Committee. Cells were cultured on laminin-plated glass coverslips in 35-mm tissue culture wells. Cells were maintained in medium 199 supplemented with 5 mmol/L each of carnitine, creatine, and taurine and 100 µg/mL penicillin–streptomycin in a 5% CO2 incubator. Heart cells were infected 2 to 3 hours after plating by adding 5 to 20 µL of adenoviral vector stock (
1011 to 1012 viral particles/mL) directly into culture wells. Virus containing media was aspirated after 4 hours and replaced with fresh serum-free media. Human embryonic kidney (HEK 293) cells were maintained in supplemented DMEM. HEK 293 cells cultured on glass coverslips in 60-mm tissue culture dishes were transiently transfected with 8 µg each of CaV channel subunits and AKAP-79 cDNA as indicated.
Electrophysiology
Whole-cell recordings were acquired at room temperature using an EPC8 or EPC10 patch clamp amplifier controlled by PULSE software. Micropipettes pulled from thin-wall borosilicate glass capillaries were filled with internal solution containing (mmol/L): 135 CsMeSO3, 5 CsCl2, 5 EGTA, 1 MgCl2, 4 ATP, and 10 Hepes (pH 7.3). Pipette resistances were typically 2 to 3 M
and compensated 50% to 70%. For heart cells, sealing and initial break-in to the whole-cell configuration was performed in external solution containing (mmol/L): 138 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 0.33 NaH2PO4, 10 Hepes, and 10 glucose (pH 7.4). Heart cell recording solution contained (mmol/L): 155 N-methyl-D-glucamine aspartate, 5 BaCl2, 10 4-aminopyridine, and 10 Hepes (pH 7.4). HEK 293 cell recording solution contained (mmol/L): 140 Et4N MeSO3, 5 BaCl2, and 10 Hepes (pH 7.4). Currents were sampled at 25 kHz and filtered at 5 or 10 kHz. Leak and capacitive transients were subtracted using a P/8 protocol.
Confocal Microscopy
Images of heart cells expressing β-GFP subunits were acquired with an Olympus Fluoview laser scanning confocal microscope using the 488-nm argon laser line for excitation.
Saturable Modulation Modeling
The basal whole-cell current density is related to microscopic channel gating parameters by: 
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where J is the whole-cell current amplitude, N is the number of channels, i is the unitary current, and Po is the basal single-channel open probability.
On exposure to PKA, the resulting increase in whole-cell current reflects the sum of currents from channels that are modulated, as well as nonmodulated channels. This can be expressed as:
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where JPKA is the whole-cell current amplitude after PKA activation, and Fmod is the fraction of channels modulated by PKA. Modulation, defined as the fold increase in current amplitude after PKA activation, is obtained by dividing Equation 2 by Equation 1 yielding:
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We can define a critical current density (Jcritical) as the maximum current at which all channels are sensitive to PKA modulation. For J
Jcritical, all of the channels are upregulated on PKA activation, and, hence, Fmod=1. However, for J>Jcritical we have,
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Substituting Equation 4 into Equation 3 yields,
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We fit the scatter plots in Figure 4C to Equation 5 with Jcritical and Po,PKA/Po as free parameters.
Data and Statistical Analyses
Data were analyzed off-line using PULSEFIT (HEKA Electronics) and Microsoft Excel software. Data are presented as means±SEM. Statistically significant differences between mean values (P<0.05) were determined by 1-way ANOVA, followed by pairwise comparisons using the Newman–Keuls procedure.
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Results |
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Difficulty in Heterologously Reconstituting PKA Modulation of Recombinant ICa,L
Many of the prevailing theories of how CaV1.2 phosphorylation by PKA leads to channel upregulation come from experiments on recombinant channels expressed in heterologous systems. A key drawback has been that many reports of successful reconstitution27,32–34 have not been universally reproducible.25,26,28,35 Nevertheless, if it were possible to reliably obtain robust PKA modulation of CaV1.2 channels in heterologous cells, this would provide a straightforward means to determine the role of CaVβs in this regulation. Accordingly, we initially focused efforts to reconstitute PKA modulation of recombinant CaV1.2 channels expressed in human embryonic kidney (HEK) 293 cells.
As a baseline for evaluating HEK 293 responses, we determined the native profile of PKA modulation in guinea pig heart cells. In these myocytes, application of 1 µmol/L forskolin invariably resulted in a substantial increase in whole-cell current (Figure 1A) that developed rapidly (Figure 1B). On average, the whole-cell current elicited by a 0-mV test pulse was increased more than 3-fold by forskolin (Figure 1I; fold increase=3.8±0.7; n=5). Could we obtain a similar robust modulation of recombinant CaV1.2 channels expressed in HEK 293 cells? We first tested this on HEK 293 cells coexpressing 1C and CaVβ2b. CaVβ2b is a splice variant of CaVβ2a, which is more dominant in the heart.8 However, the C termini (which contain the PKA phosphorylation sites) are identical between the 2 splice variants.15 In sharp contrast to our observations in guinea pig heart cells, application of 1 µmol/L forskolin did not increase ICa,L reconstituted in HEK 293 cells (Figure 1C, 1D, and 1I). This absence of modulation is consistent with what has been reported previously by several groups.25,26,28,35
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A leading concept is that an A kinase anchoring protein (AKAP) is a crucial missing ingredient whose reintroduction permits successful reconstruction of PKA-mediated upregulation of heterologously expressed CaV1.2 channels.27,36 We tested this idea in HEK 293 cells coexpressing 1C/β2b and AKAP-79. Surprisingly, even with AKAP-79, we could elicit no response on recombinant ICa,L with forskolin (Figure 1E, 1F, and 1I). This finding is in agreement with a previous result obtained using recombinant channels expressed in Xenopus oocytes.35 Finally, we sought to reproduce conditions similar to the original report that suggested the importance of CaVβ2 phosphorylation to PKA modulation.22 In that study, it was suggested that phosphorylation of β2 gained prominence in functional modulation when channels were reconstituted with a pore-forming 1C subunit truncated at residue 1905 (1C
1905). Therefore, we tested whether channels reconstituted with 1C1905/β2b could possibly reconstitute PKA modulation in HEK 293 cells. However, we observed no effect of forskolin on 1C1905/β2b channel currents (Figure 1G, 1H, and 1I).
We conclude from these results that successful reconstitution of PKA modulation of recombinant CaV1.2 channels in heterologous cells remains an elusive target. Indeed, to date, there is no report of PKA modulation of ICa,L being reconstructed heterologously in the robust manner evident in the guinea pig heart (Figure 1A and 1B). The results emphasize the ambiguities associated with gleaning mechanistic insights into functional PKA modulation of ICa,L from heterologous cells. Accordingly, we sought to pursue our studies in the context of heart cells where modulation is reproducibly robust.
Eliminating Phosphorylation Sites in β2a Does Not Diminish PKA Modulation
The β2 subunit in the heart is phosphorylated by activated PKA.23,37 We first sought to test the idea that β2a phosphorylation is important for PKA-mediated increase in ICa,L. As a prelude to these experiments, we established baseline CaV1.2 channel properties in guinea pig heart cells overexpressing wild-type β2a-GFP (Figure 2). The presence of the GFP tag permitted unambiguous identification of cells robustly expressing the exogenous β2a subunit. In cells expressing wild-type β2a-GFP, the fluorescence signal was localized to the sarcolemma and transverse tubular membrane (Figure 2A), consistent with the posttranslational palmitoylation known to occur on this CaVβ subunit.38 Overexpression of β2a-GFP in guinea pig heart cells resulted in a dramatic increase in basal current density (Figure 2B and 2I) across the entire relevant voltage range (Figure 2C) compared with control cells expressing GFP alone. Moreover, the current density versus voltage (J-V) relationship showed a hyperpolarizing shift compared with control (Figure 2C). These results are consistent with previous observations in rat8,39 and feline heart cells.40 Could channels reconstituted with exogenous β2a be modulated by PKA in the native cardiac environment? Indeed, exposure of heart cells overexpressing β2a-GFP to forskolin consistently resulted in an increase in whole-cell current amplitude. Importantly, however, the magnitude of the response was markedly lower compared with the degree of modulation observed in control heart cells expressing GFP alone (Figure 2D and 2J). Nevertheless, the residual modulation seen in the β2a-modified channels provided an opportunity to investigate whether direct phosphorylation of β2a was important for PKA regulation of ICa,L.
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). D, Exemplar whole-cell Ba2+ currents in a guinea pig cell expressing β2a-GFP before (black trace) and after (red trace) exposure to 1 µmol/L forskolin. E, Confocal image of a guinea pig heart cell expressing β2a,AAA-GFP. F, Exemplar whole-cell currents from guinea pig cell expressing β2a,AAA-GFP. G, Population J-V plots for heart cell expressing GFP alone (cyan trace) or β2a,AAA-GFP (
). H, Exemplar whole-cell currents before (black trace) and after (red trace) 1 µmol/L forskolin in a heart cell expressing β2a,AAA-GFP. I, Bar chart showing relative effects of β2a-GFP and β2a,AAA-GFP on basal current density in guinea pig myocytes. One-way ANOVA indicated a statistically significant difference among treatment groups [F(2,13)=18.6; P<0.001]. *Significantly different from GFP-infected cells based on the Newman–Keuls procedure (P<0.05). J, Bar chart showing relative magnitude of PKA modulation in control cells expressing GFP compared with cells expressing β2a-GFP and β2a,AAA-GFP, respectively. One-way ANOVA indicated a statistically significant difference among treatment groups [F(2,14)=13.17; P<0.001]. *Significantly different from GFP-infected cells based on the Newman–Keuls procedure (P<0.05).To achieve this, we generated an adenovirus encoding a mutated β2a (termed β2aAAA) in which 3 confirmed PKA phosphorylation sites (Ser459, Ser478, and Ser 479)23 are converted to alanines. Expression of β2aAAA-GFP in guinea pig heart cells resulted in fluorescence in the sarcolemmal and transverse tubular membrane, similar to wild-type β2a-GFP (Figure 2E). Moreover, β2aAAA-GFP enhanced basal current density (Figure 2F, 2G, and 2I) and shifted the voltage-dependence of channel activation (Figure 2G) to virtually the same extent as observed with wild-type β2a-GFP. Most importantly, the fold increase in current amplitude in response to forskolin was unchanged between β2aAAA-GFP and wild-type β2a-GFP channels (P=0.13, by unpaired Student’s 2-tailed t test) (Figure 2H and 2J). This result shows that direct phosphorylation of β2a subunits is not necessary for any portion of ICa,L upregulation by PKA.
Impact of β Subunit Identity on PKA Modulation of CaV1.2 Channels
The identity of the β subunit constituent of assembled CaV1.2 channels could still play a major role in dictating the extent of channel regulation by activated PKA, even though we find that direct phosphorylation of β2 is not necessary. To investigate this, we compared the degree of PKA modulation among heart cells overexpressing the four distinct CaVβ (β1 to β4) subunits (Figure 3). By comparison with control heart cells expressing GFP alone, each of the β subunits caused a significant increase in whole-cell current amplitude (Figure 3A and 3B), as indicated by ANOVA (F=15.03; df=4,42; P<0.001) and pairwise comparisons using the Newman–Keuls procedure. Moreover, in isochronal experiments, the increase in current amplitude recorded with β2a-GFP expression was significantly higher than that achieved with the other CaVβ subunits (Figure 3B). Why does β2a increase basal current density to a larger extent than other β subunits? We showed previously that all 4 β subunits expressed in the heart increased the maximum gating charge (Qmax), suggesting that they increase the number of channels with moveable voltage sensors in the plasma membrane.8 This phenomenon provides a baseline mechanism for how all overexpressed CaVβs increase whole-cell current in the heart. Beyond the increase in the number of channels, β2a was unique in also increasing the single channel open probability (by more than 2.5-fold compared with control), principally by ablating null sweeps.8 This unique action of β2a on Po provides a simple rationale for its larger effect on basal whole-cell current amplitude compared with other β isoforms.
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We next determined how each overexpressed CaVβ subunit affects PKA modulation of endogenous CaV1.2 channels. To facilitate visual comparison of the relative effects, in Figure 3C we show normalized average basal current traces for each condition (black traces) and the fold increase obtained with 1 µmol/L isoproterenol (red traces). In control cells, the magnitude of the response to 1 µmol/L isoproterenol was essentially the same as obtained with 1 µmol/L forskolin. By comparison with the degree of modulation seen in control, all cells expressing exogenous β subunits exhibited a diminished upregulation of current, albeit to different extents (Figure 3C and 3D). Strikingly, channels reconstituted with β2a exhibited the weakest modulation. This result is consistent with the notion that the higher basal single-channel Po conferred by β2a results in a diminished dynamic range of PKA modulation. Moreover, this result serves as a dramatic demonstration of how β subunit identity can greatly impact the magnitude of the PKA response in CaV1.2 channels even without involvement of direct phosphorylation.
A Limiting Resource for PKA Modulation of CaV1.2 Channels
It was worthwhile to consider mechanisms that could account for the smaller degree of PKA-mediated upregulation of ICa,L in cells overexpressing the other β isoforms (other than β2a) compared with control. Unlike the situation with β2a, these could not be rationalized by a diminished dynamic range of modulation because single-channel experiments did not show that these β subunits increased Po relative to control.8 To understand the mechanistic bases of this phenomenon we considered 2 different ways in which it could arise, with the common assumption that overexpressing CaVβs increases the number of channels in the sarcolemma (Figure 4A). First, all channels in the membrane could be upregulated uniformly in response to PKA activation but at a diminished overall level compared with the control condition (Figure 4A, uniform modulation scenario). This could occur if, for example, individual β subunits conferred subtly distinct configurations to the CaV1.2 channel that lead to intrinsic differences in how the channel responded to activated PKA. A prediction of this scenario is that the degree of channel modulation would be fairly independent of basal current density, and a scatter plot of the 2 variables would be expected to yield an approximately constant value (Figure 4B). This scenario also implies a significant functional reserve for PKA modulation because additional channels can be accommodated without appreciable loss of regulation. Alternatively, the diminished modulation could be explained by a circumstance in which there are only a limited number of permissive sites on the membrane where PKA-mediated upregulation of ICa,L can occur (Figure 4A, saturable modulation scenario). Here, increasing the number of channels in the membrane would quickly saturate these regulation-permissible loci. Hence, channels in excess of these sites would be unresponsive to PKA, thereby diluting the overall effect and resulting in a diminished whole-cell response when compared with control. This scenario predicts that the degree of channel modulation would vary inversely with the basal current density, and a scatter plot of the 2 variables would be expected to adopt a hyperbolic relationship (Figure 4B). A corollary of this scenario is that there is essentially no functional reserve for PKA upregulation of ICa,L in the heart.
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) to high Po mode (
), β2a-GFP (
), β3-GFP (
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To distinguish between these 2 possibilities, we generated a scatter plot of the degree of channel modulation versus basal current density for individual experiments. This strategy revealed an unmistakable inverse relationship between the 2 variables (Figure 4C). Moreover, the data were well fit by the following analytic expression that was derived assuming a saturable modulation model (see Materials and Methods):
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where J is the basal current density, Jcritical is the maximum basal current density at which all channels are PKA sensitive, Po is the basal open probability, and Po,PKA is the open probability of PKA-modulated channels. A least-squares fit of the data using the numeric solver in Excel and with Jcritical and Po,PKA/Po as free parameters converged at values of 6.9 pA pF–1 and 3.9, respectively. Overall, these results indicate a limiting resource for PKA modulation of ICa,L that results in a limited functional reserve for this physiological regulation in the heart. How prevalent is this design feature among different species? To address this, we performed similar experiments in adult rat heart cells. Control rat heart cells expressing GFP displayed larger basal current density and a lower magnitude of PKA modulation compared with their guinea pig counterparts (Figure 4C). As previously shown,8 overexpressing all CaVβs resulted in an increased basal current density (Figure 4C). Similar to the guinea pig data, there was an inverse relationship between the strength of channel modulation and basal current density (Figure 4C). Least-squares fit of the data yielded values for Jcritical and Po,PKA/Po of 6.7 pA pF–1 and 3.4, respectively. The similar values for the 2 parameters obtained in different species provide 2 important insights. First, it demonstrates that the limited functional reserve in PKA regulation of CaV1.2 channels is a rather conserved design principle in the heart. Second, it suggests that the lower degree of PKA modulation observed in rat versus guinea pig heart can be explained by differences in the fraction of unresponsive channels, rather than intrinsic distinctions in channel properties between the 2 species.
One possibility, based on our use of isoproterenol in these studies, was that β-adrenergic receptors represented the limiting resource for PKA modulation of CaV1.2 channels in the heart. Previous studies have identified a macromolecular complex that includes β-adrenergic receptors and CaV1.2 channels in neurons.41 We investigated this possibility by using 5 µmol/L forskolin to upregulate CaV1.2 channels in guinea pig heart cells overexpressing β2b (Figure 5). The degree of channel modulation with forskolin displayed an inverse relationship with the basal current density and agreed closely with the curve generated using isoproterenol as the activating agent. This result suggests that the limiting resource for PKA modulation of CaV1.2 channels in the heart lies beyond the β-adrenergic receptors.
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The use of β2b in these experiments is noteworthy because it is predominant over β2a as the endogenous β in rodent and human heart.8,42 The fact that overexpressed β2b results in channels that are robustly modulated in a manner conforming to the saturable modulation model is entirely consistent with the ample regulation of native channels. By contrast, if β2a were the dominant isoform expressed endogenously in the heart, our results would predict a much smaller PKA upregulation of native cardiac L-type channels than is commonly observed.
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Discussion |
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Our studies provide 2 novel insights into regulation of cardiac L-type CaV channels by activated PKA. First, we find that the identity of the CaVβ subunit assembled in the mature CaV1.2 channel can have a major impact on the magnitude of ICa,L upregulation by PKA. However, the most important property of a β subunit that influences the strength of modulation is its effect on basal channel Po, rather than its being a substrate for PKA phosphorylation. Second, we find that that there is no excess capacity for PKA upregulation of CaV1.2 channels in the heart. We discuss the implications of these unexpected results in relation to previous studies.
Importance of the Experimental System in Studying PKA Modulation of ICa,L
An essential element of our study is that we used adult heart cells to probe molecular mechanisms of PKA upregulation of ICa,L. By contrast, most previous studies on this subject have used recombinant CaV1.2 channels expressed in heterologous cells. Our approach offers a number of key advantages. First, historically, reconstitution of PKA upregulation of calcium currents in heterologous cells has been fraught with inconsistency, with early reports of success32–34 not universally reproducible.25–28 An influential concept is that coexpressing AKAPs permits successful heterologous reconstruction of PKA modulation of ICa,L,27,36 albeit in a much less robust manner than observed in the heart. Unfortunately, we were unable to demonstrate PKA modulation of ICa,L in HEK 293 cells even with coexpression of AKAP-79, in agreement with a previous observation in Xenopus oocytes.35 Other theories have been advanced to explain the inability to readily observe acute PKA-mediated upregulation of recombinant ICa,L in heterologous cells. For example, it has been suggested that recombinant CaV1.2 channels may be basally phosphorylated and, hence, already upregulated in heterologous cells.25,43 This view is contradicted by findings that forskolin-induced phosphorylation of Ser1928 of CaV1.2 1 can be demonstrated in intact HEK 293 cells.29 More recently, it has been suggested that AKAP-79 also localizes the phosphatase calcineurin, which so efficiently dephosphorylates the channel that it prevents observation of PKA-induced ICa,L upregulation in HEK 293 cells.44 However, even when recombinant CaV1.2 channels are coexpressed with AKAP-18, which does not bind calcineurin, the strength of ICa,L modulation reported (18% average increase) falls well short of what is seen in the heart and is inconsistent.36 Overall, the multitude of conflicting data regarding the feasibility of reconstituting PKA modulation of recombinant CaV1.2 channels, together with the diverse hypotheses proposed to explain the different viewpoints constitutes a challenge in reaching an articulated consensus. We conclude that heterologous reconstitution of robust PKA modulation of ICa,L remains a challenging prospect. Unfortunately, this generates much uncertainty regarding prevailing notions of the molecular mechanisms linking PKA phosphorylation to ICa,L modulation, most of which have been based on heterologous reconstitution studies that have proven notoriously difficult to reproduce.
A second advantage of our study is the reliance on endogenous CaV1.2 1C subunits. There is some ambiguity regarding the relative prevalence of different configurations of cardiac 1C subunits in the heart and their potential contribution to PKA regulation of ICa,L. Although the 2171-aa primary sequence of cardiac 1C predicts a 240-kDa protein, Western blot and antibody mapping studies indicate that a substantial fraction of 1C in the heart may exist as a 190-kDa protein, truncated in the vicinity of amino acid residue 1905. Nevertheless, the 1C C terminus was detected in a comparable ratio to the main body in intact heart cells by immunofluorescence,45 suggesting a third possible configuration of CaV1.2 channels in which a posttranslationally cleaved 50-kDa C terminus is noncovalently associated with the truncated 190-kDa 1C subunit.45,46 It has recently been shown that the truncated CaV1.2 C terminus can function as a transcription factor.47 Notably, the putative posttranslational proteolysis of cardiac 1C is absent in heterologous systems.48 Nevertheless, the dominant configuration of 1C in the heart is important because putative sites necessary for PKA modulation of the channel (such as Ser1928 and an AKAP binding site) are located downstream of the presumed proteolytic site.49 Here, by confining our studies to endogenous 1C, we circumvent uncertainties about the functionally dominant configuration of the 1C C terminus in the heart.
Role of CaVβs in PKA Modulation
In intact myocytes, the β2 subunit is phosphorylated in response to β-adrenergic receptor stimulation.23,37 The sites of phosphorylation were elegantly mapped to 3 serine residues (Ser459, Ser478, and Ser479) located in the nonconserved C-terminal domain of β2a.23 Subsequently, it was reported that phosphorylation of β2a on residues Ser478 and Ser479 was critical to PKA modulation of recombinant channels but only within a narrowly defined circumstance: when channels were reconstituted with a 1C truncated at residue 1905.22 In contrast to this previous report, we could not reconstitute PKA-mediated upregulation of currents reconstituted with 1C1905/β2 channels in HEK 293 cells (Figure 1), preventing independent verification of an essential role for β2a phosphorylation in this setting. However, by comparing PKA modulation of ICa,L in heart cells expressing either wild-type β2a or a phosphorylation-deficient mutant, β2a,AAA, we show that phosphorylation of β2a is not necessary for ICa,L upregulation in the heart. A recent article reported on PKA regulation of ICa,L in heart cells overexpressing a double mutant β2a[S478A,S479A]. Similar to our observations, expression of β2a[S478A,S479A] in heart cells lead to a higher basal current density and a diminished PKA response compared with control cells.50 However, in that study, comparative experiments with wild-type β2a were not performed, leaving open the interpretation that the diminished response was attributable to loss of phosphorylation in β2a[S478A, S479A]. Here, by explicitly comparing CaV1.2 channel modulation in cells expressing wild-type β2a versus β2a,AAA, we can definitively conclude that β phosphorylation is not necessary for PKA regulation of ICa,L. This conclusion is further consolidated by the new finding that channels reconstituted with other β-subunit isoforms that do not contain the β2a phosphorylation sites are, nevertheless, robustly upregulated by PKA. Overall, the strength of our conclusions is critically dependent on the assumption that overexpressed exogenous CaVβs are predominant in sarcolemmal channels compared with endogenous CaVβs. This assumption appears justified for a number of reasons. First, exogenous CaVβ subunits are overexpressed to a level almost 10-fold greater than their endogenous counterparts.8 Second, whole-cell current kinetic properties change to reflect the properties of the expressed CaVβ subunit (such as slowed inactivation in cells expressing β2a or β4). Third, it has been directly demonstrated that competitive exchange of acutely injected β-subunit protein can occur with β subunits present in channels preexisting in the plasma membrane in Xenopus oocytes.51 Finally, we simulated (data not shown) the expected PKA response if currents recorded in β2a-expressing cells were made up of a mixture of pure β2a channels and varying fractions of endogenous channels. The simulations indicate that if the full cadre of endogenous channels were present alongside pure β2a channels, the degree of PKA modulation expected would be substantially higher than what was actually observed. This was the case even when it was assumed that only 20% of the endogenous β channels remained, with the remainder having been exchanged with β2a.
Even though direct phosphorylation of CaVβs may not be necessary for channel modulation, we, nevertheless, found that the identity of CaVβ in the channel complex could profoundly affect the magnitude of PKA regulation of ICa,L. Specifically, channels assembled with β2a displayed a significantly diminished PKA response compared with other β isoforms. We interpret this result in light of previous single-channel experiments that demonstrate the prevalence of distinct gating modes in native CaV1.2 channels: mode 0 (null sweeps), mode 0a (brief, infrequent openings), mode 1 (frequent, millisecond openings), and mode 2 (long-lasting openings).3,24,52 Under basal conditions, channel gating is dominated by mode 0/0a and mode 1 openings with rare excursions into mode 2. Activated PKA increases channel Po by reducing the frequency of mode 0/0a while increasing the prevalence of mode 1 and 2 gating.24 Native cardiac CaV1.2 channels reconstituted with β2a displayed a 3- to 4-fold higher basal Po compared with channels incorporating other β subunits, principally because of an ablation of null gating, as well as enhanced mode 1 gating in active sweeps.8,42 This result predicts that β2a channels would uniquely have a substantively lower response to activated PKA because of a diminished dynamic range of modulation, consistent with our observations. A relevant question is whether β2a overexpression in the heart increases channel Po via an effect on basal channel phosphorylation either through PKA or calmodulin kinase II. Although this scenario is possible, it appears unlikely for 2 reasons. First, the basal single-channel signature of β2a-expressing channels is devoid of the long-lasting mode 2 openings that characterize channels upregulated by PKA or calmodulin kinase II.8,24,53 Second, the closely related splice variant, β2b, does not similarly increase single-channel Po. It is difficult to envision why β2a would uniquely increase basal channel phosphorylation, whereas the closely related β2b does not. The most parsimonious explanation is that β2a increases channel Po via a process independent of basal channel phosphorylation. Indeed, the majority of β2a effects on single-channel gating could be modeled by a reduction in the microscopic propensity for inactivation.8 Hence, it is tempting to speculate that the unique aspect of β2a that leads to an increased single-channel Po is the posttranslational palmitoylation that enables it to autonomously target to the plasma membrane, and also confer slow macroscopic inactivation kinetics to calcium currents.15,38,54
Our results also have bearing on a recent study in which PKA modulation of a dihydropyridine insensitive full-length 1C, 1C(D-), was compared with a truncated mutant, 1C(D-)1905, following adenoviral-mediated gene transfer in guinea pig myocytes.50 In that study, it was found that 1C(D-)1905 displayed significantly weaker PKA-mediated upregulation compared with 1C(D-) or 1C(D-)S1928A channels. This was interpreted as indicating a necessary role for the 1C C terminus in PKA modulation. However, truncating the 1C C terminus is known to increase the basal channel Po.55 Hence, an alternative interpretation is that 1C(D-)1905 shows weaker modulation because of a smaller dynamic range for modulation, similar to our proposal here for native channels reconstituted with β2a.
Limited Functional Reserve for PKA Modulation of ICa,L in the Heart
A surprising finding from our study is that there is a limited functional reserve for PKA modulation of CaV1.2 channels in the heart. This finding has important implications. First, this result may bear directly on the difficulties in reconstituting PKA regulation in heterologous cells. Efforts in this regard have been driven by the fundamental assumption that PKA upregulation of ICa,L is an intrinsic property of CaV1.2 channels that is unfailingly triggered by the appropriate posttranslational modification (phosphorylation) of specific residues. Our results challenge this basic assumption. Even in the heart, where PKA modulation is most robust and reproducible, our analyses suggest that a substantial fraction of CaV1.2 channels behave as if they cannot be upregulated by activated PKA. What is the limiting resource for PKA modulation of ICa,L in the heart? The answer could fall in 1 of 2 main categories: (1) conditions that prevent appropriate phosphorylation of the channel or (2) conditions that prevent an appropriately phosphorylated channel from switching into a high-activity gating mode. Possible candidates for the first category include: absence of an AKAP from some CaV1.2 channel complexes; or targeting of channels to locations with unfavorable local environments, for example, with high phosphodiesterase or phosphatase activities. Implicit in the second category is the idea that appropriate phosphorylation of the channel alone is not sufficient for enhancing channel activity but that actors outside the core channel complex (comprised of 1, β, and 2 subunits) are also essential for functional transduction. If such extrachannel players were cardiac-specific, this would provide a nice rationale for the discrepant PKA regulation of ICa,L in heart versus heterologous cells.56 We cannot distinguish between these 2 categories based on our present data. However, the experimental paradigm used here presents a unique system to explore such questions in future studies.
Second, an enigmatic feature of PKA modulation of cardiac ICa,L is that the extent of reported regulation can differ substantially among hearts from different species. For example, PKA-induced upregulation of ICa,L is typically greater in guinea pig compared with rat cardiac myocytes.5 The mechanistic bases for such differences are unknown. Our results suggest that this variation may be predominantly explained by relative differences in the fraction of unmodulated channels, rather than intrinsic distinctions in CaV1.2 channel properties between the two species. Differences in the expression pattern of CaVβ subunits could also potentially contribute. It will be interesting to determine whether such a mechanism can also explain the generally lower degree of PKA modulation of CaV1.2 channels observed in other tissues such as smooth muscle and neurons compared with the heart within the same organism. Finally, our results suggest that either an increase in the fraction of unmodulated channels, or alterations in the complement of β subunits expressed,42 could be contributing mechanisms to the degraded PKA modulation of cardiac ICa,L typically observed in heart failure.5,57
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Acknowledgments |
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We thank Robert Kass, Timothy Kernan, Steven Marx, and Ming Zhou for helpful comments on the manuscript.
Sources of Funding
This work was supported by grants from the NIH (to H.M.C) and MERIT award 5R37HL076795 from NHLBI (to D.T.Y).
Disclosures
None.
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Footnotes |
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This manuscript was sent to David Eisner, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received January 10, 2008; revision received February 21, 2008; accepted March 12, 2008.
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