Molecular basis for the modulation of native T-type Ca²⁺ channels in vivo by Ca²⁺ /calmodulin-dependent protein kinase II

Ang II receptor activation increases cytosolic Ca²⁺ levels to enhance the synthesis and secretion of aldosterone, a recently identified early pathogenic stimulus that adversely influences cardiovascular homeostasis. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is a downstream effector of the Ang II–elicited signaling cascade that serves as a key intracellular Ca²⁺ sensor to feedback-regulate Ca²⁺ entry through voltage-gated Ca²⁺ channels. However, the molecular mechanism(s) by which CaMKII regulates these important physiological targets to increase Ca²⁺ entry remain unresolved. We show here that CaMKII forms a signaling complex with α_1H T-type Ca²⁺ channels, directly interacting with the intracellular loop connecting domains II and III of the channel pore (II-III loop). Activation of the kinase mediated the phosphorylation of Ser1198 in the II-III loop and the positive feedback regulation of channel gating both in intact cells in situ and in cells of the native adrenal zona glomerulosa stimulated by Ang II in vivo. These data define the molecular basis for the in vivo modulation of native T-type Ca²⁺ channels by CaMKII and suggest that the disruption of this signaling complex in the zona glomerulosa may provide a new therapeutic approach to limit aldosterone production and cardiovascular disease progression.

Abnormal intracellular Ca²⁺ homeostasis is a common feature of cardiovascular disease (1, 2). In the human failing heart, arrhythmogenic remodeling is characterized by dysregulated sarcoplasmic reticulum Ca²⁺ uptake and release, enhanced Na-Ca²⁺ exchange activity, and increased activity of L-type high voltage–activated (HVA) Ca²⁺ channels (α_1C) (3–5) in cardiomyocytes. Central to these pathologies is an increase in adrenergic tone and a marked activation of the renin-angiotensin aldosterone axis. The importance of aldosterone as an early-onset pathogenic stimulus has been highlighted by recent findings. Following myocardial infarction, inappropriate mineralocorticoid receptor activation increases L-type Ca²⁺ channel density before the onset of structural hypertrophy (6), and chronic levels of circulating aldosterone correlate directly with the density of L-type HVA Ca²⁺ channels in ventricular myocytes that are devoid of morphological change (7). In addition, in the setting of high Na⁺ intake, the progression to left-ventricular hypertrophy and failure is advanced by an elevation in aldosterone levels that stimulates perivascular fibrosis (8–10). Thus, long recognized to regulate Na⁺ and K⁺ balance, blood volume, and blood pressure, aldosterone has a newly appreciated adverse influence on cardiovascular homeostasis.

Aldosterone is synthesized and secreted from the adrenal zona glomerulosa (ZG) cell principally in response to Ang II, ACTH, and physiological increases in extracellular potassium (11). These secretagogues depolarize the ZG cell and/or potentiate Ca²⁺ channel activity (12). Aldosterone production is Ca²⁺ dependent, and low voltage–activated (LVA), T-type Ca²⁺ channels provide the Ca²⁺ that is necessary to sustain its stimulated production (13–15).

Voltage-dependent Ca²⁺ channels change electrical signals into biochemical events. But, because this conversion is adjustable, via changes in channel activity and density, Ca²⁺ channels also can exert additional controls on Ca²⁺-dependent cellular processes. In the heart, L-type Ca²⁺ channels use Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) to adjust signal transmission by inducing a gating change that is characterized by frequent channel openings (16). CaMKII expression is increased in structural heart disease (17), and inhibition of its activity reduces Ca²⁺ current and ameliorates dysfunction (18). In the ZG cell, CaMKII induces a gating shift of α_1H T-type Ca²⁺ channels that enables more channels to open at hyperpolarized potentials, enhancing Ca²⁺ entry and thus stimulating aldosterone production (19). Although CaMKII regulates both α_1C HVA (16, 20, 21) and α_1H LVA channels (19, 22, 23), the molecular basis for these effects in vitro and in vivo remains unresolved. Here we analyzed the interaction of CaMKII with α_1H T-type Ca²⁺ channels. Our data show that α_1H channels use tethered CaMKII as a Ca²⁺ sensor to dynamically regulate channel activity. Moreover, our data indicate that α_1H channels are regulated robustly by protein phosphorylation in situ and in vivo, identifying these channels for the first time to our knowledge as authentic physiological targets of kinse activity.

LVA channels are encoded by 3 separate genes: α1G, α1H, and α1I, belonging to the Ca_v3.0 family of voltage-gated Ca²⁺ channels (24). CaMKII activation increases whole-cell and single-channel currents mediated by α_1H channels by inducing an approximately 12-mV hyperpolarizing shift in the half-activation potential for channel opening. This change in activation gating mediated by CaMKII depends on the intracellular loop (residues 1019–1293) connecting channel transmembrane domains II and III and can be abrogated by a Ser1198-to-Ala point mutation (23). Therefore, to characterize the interaction of CaMKII with α_1H channels, we began by investigating the binding of purified recombinant CaMKII to the II-III loop, which was expressed as a bacterial fusion protein (see Methods). After the proteins were mixed (8 hours, 4°C), bound kinase was separated using glutathione-sepharose. Binding was quantified against purified recombinant CaMKII protein standards (0.25–1 pmol) in immunoblots using a pan-CaMKII–specific antibody. Because CaMKII displays differing activity states, we evaluated binding under conditions that promoted each configuration. We activated CaMKII with Ca²⁺/calmodulin (Ca²⁺/CaM) to release the substrate-binding domain of the kinase from autoinhibitory contacts made in the basal state (25–27) or included Ca²⁺/CaM and ATP in the binding buffer to allow the transphosphorylation of Thr287 between adjacent kinase monomers within the dodecameric holoenzyme (28). Autophosphorylation sustains kinase activity in the absence of Ca²⁺ elevation and also exposes additional sites for protein-protein interactions (29, 30). Notably, inactive kinase, in its autoinhibited conformation, bound robustly to the channel loop (0.14 ± 0.03 pmol/pmol loop at 20 nM CaMKIIγ_C; n = 5) (Figure 1A). This binding was specific to the loop and could not be attributed to the agarose bead or to the N terminus of the glutathione-S-transferase (GST) fusion protein. Moreover, syntide-2, a CaMKII substrate modeled after the CaMKII phosphorylation site of glycogen synthase, could not compete for binding of the inactive kinase at either 50 or 500 μM (Figure 1D), suggesting that the substrate-binding domain of CaMKII is not mediating effector contact. Surprisingly, activation of CaMKII by Ca²⁺ (0.5 mM), CaM (5 mM), and ADP (0.5 mM) had no significant effect on the level of binding (0.12 ± 0.02 pmol/pmol loop; n = 5; NS). Nonetheless, the binding of active kinase was effectively competed for by syntide-2. Syntide-2 reduced the binding of active kinase by 38% ± 4% (n = 4; P < 0.05) and 89% ± 9% (n = 3; P < 0.05) at 50 μM and 500 μM, respectively (Figure 1D). Taken together these data suggest that the inactive kinase interacts with the α_1H II-III loop at a primary contact site that lies outside of its classical substrate-binding domain.

Figure 1

CaMKIIγ_C binds to the α_1H II-III loop and is released by loop phosphorylation. (A–C) Binding of purified recombinant CaMKIIγ_C (5 pmol/250 μl) to bead-bound GST-(II-III) loop fusion proteins (2 pmol). Wild-type α_1H II-III loop (A), RAE(S¹¹⁹⁸)A–α_1H II-III loop (B), or α_1G II-III loop (C) under autoinhibited (basal), activated (Ca²⁺/CaM + ADP), or autophosphorylated (Ca²⁺/CaM + ATP) conditions. Upper panels: immunoblots of bound kinase detected by anti-CaMKII antibody; 0.25 pmol standard for comparison. Equal fusion loading was verified independently in parallel runs (see Methods), and anti-GST detection is shown below. Histograms: quantified mean binding data ± SEM for 5 studies. *P < 0.05, Ca²⁺/CaM + ATP versus basal and Ca²⁺/CaM + ADP (Kruskal-Wallis ANOVA on Ranks, Dunn’s method). (D) Competition for autoinhibited or Ca²⁺/CaM + ADP–activated CaMKII/α_1H II-III loop by syntide-2. *P < 0.05 (ANOVA, Student-Newman-Keuls method). (E and F) Time course of 4°C CaMKIIγ_C dissociation and loop phosphorylation following activation of autoinhibited CaMKIIγ_C prebound to GST–α_1H II-III loop fusion proteins [wild type vs. RAE(S¹¹⁹⁸)A]. Activators, Ca²⁺/CaM + ATP (0.5 mM/5 μM + 0.5 mM), added at t = 0. (E) Activated-kinase binding determined from quantified immunoblots expressed relative to preactivation values. (F) Phosphorylation stoichiometry of GST–α_1H II-III loop fusions determined from ³²P incorporation. *P < 0.05, wild type binding at 5 or 10 minutes versus 0 minutes (ANOVA, Student-Newman-Keuls method). Inset: 30°C. Data are expressed as mean ± SEM (n = 3).

We also examined the binding of catalytically active kinase by replacing ADP with ATP in the activation buffer. ATP (0.5 mM) reduced CaMKII binding by more than 60% to 0.05 ± 0.001 pmol/pmol (n = 5), indicating that either CaMKII autophosphorylation or CaMKII phosphorylation of the II-III loop disrupts binding (Figure 1A). Therefore, we examined the binding of CaMKII to a mutated II-III loop in which the major CaMKII phosphorylation site, Ser1198 (23), was mutated to Ala (Figure 1B). The mutated loop bound similar amounts of inactive CaMKII compared with the wild-type loop (0.15 ± 0.01 pmol/pmol; n = 5). Notably, CaMKII activation (Ca²⁺/CaM + ADP) or autophosphorylation (Ca²⁺/CaM + ATP) had no significant effect on this interaction (0.15 ± 0.01 pmol/pmol [ADP], 0.13 ± 0.01 pmol/pmol [ATP]; n = 5; NS). Thus, Ser1198 substrate phosphorylation appears to initiate the release of the kinase from the channel loop. As expected, we observed no evidence for the binding of inactive, active, or autonomously active CaMKII to the α_1G II-III loop fusion protein despite overexposure of our immunoblots (Figure 1C), in agreement with the lack of regulation of α_1G channels by CaMKII (22).

To corroborate the above-described findings, we preassembled complexes of inactive CaMKII with wild-type or S1198A-mutated II-III loops and followed the time course of reduction in binding upon activation of the kinase with Ca²⁺/CaM and ATP/[γ-³²P]ATP at 4°C to slow enzymatic activity. Kinase preassociated with the wild-type loop (Figure 1E) was released with a time course (T_1/2 = 2.1 minutes, for 0–5 minutes, where T_1/2 represents half-time of dissociation) that approximately paralleled the time course of phosphorylation (Figure 1F) of the loop (T_1/2 = 2.7 minutes, for 0–5 minutes). At 30°C the rate of debinding was dramatically increased (Figure 1E, inset), consistent with the increased rate of loop phosphorylation (Figure 1F, inset). Mutation of Ser1198 dramatically reduced both phosphate incorporation (73% ± 10%; n = 3; Figure 1F) and the dissociation of the kinase (94% ± 4% still bound at 10 minutes; n = 3; Figure 1E). Thus, our data show that the interaction of CaMKII with the II-III loop depends on its activity state: inactive kinase forms a stable interaction, whereas the interaction of active kinase is transient. These features are quite distinct from the long-lasting interactions of CaMKII with N-methyl- d-aspartic acid (NMDA) receptors (31–35) or ether-a-go-go channels (36, 37) that can keep the active kinase tethered in a catalytically active conformation even in the absence of Ca²⁺ elevation or autophosphorylation.

To determine whether CaMKII interacts with α_1H channels in situ, we coexpressed CaMKIIγ_C and FLAG-tagged α_1H channels in HEK 293 cells and tested for coimmunoprecipitation. Immunoblotting with a pan-CaMKII–specific antibody often revealed 2 bands of immunoreactivity that coimmunoprecipitated with CaMKIIγ_C-transfected α_1H channels: an upper band corresponding to CaMKIIγ_C and a lower band of weaker immunoreactivity, which was less consistently observed and likely to be a truncation product of the expressed kinase construct (Figure 2A). Moreover, CaMKII was not detected in the absence of an immunoprecipitating antibody, in the absence of channel or CaMKII expression, or in the immunoprecipitate of a control immunoglobulin, validating the specificity of the observed coassociation. Because the II-III loop of α_1H channels confers regulation to α_1G channels that are not modulated by CaMKII, we hypothesized that substitution of the α_1H II-III loop with the II-III loop from α_1G channels would reduce the amount of CaMKII coimmunoprecipitated. As illustrated in Figure 2A in a representative immunoblot, the chimeric channel immunoprecipitated a fraction of the CaMKII immunoprecipitated by the wild-type α_1H channel, despite equivalent levels of CaMKII expression detected in the lysate input. In 3 of 9 experiments where the efficiency of channel immunoprecipitation was equivalent, the α_1H(GII-III) chimera immunoprecipitated 33.7% ± 0.5% of the CaMKII immunoreactivity that was immunoprecipitated with the wild-type channel (Figure 2B). Thus, contact sites in the II-III loop play an important role in CaMKII binding to α_1H channels in situ. Nonetheless, because one-third of the immunoreactivity was retained by the chimeric channel, we tested for the coassociation of CaMKII with other intracellular channel domains that were expressed as GST fusion proteins. These GST fusion proteins were loaded onto glutathione-sepharose and used in in vitro binding reactions with purified CaMKII. No evidence was obtained for the interaction of CaMKII with the N terminus, the I-II loop, or the C terminus (Figure 2C). However, significant binding to the III-IV loop, representing 10–16% of the binding to the α_1H II-III loop, was observed. In principle, CaMKII binding to the III-IV loop can account in part for the residual binding of CaMKII to the α_1H(GII-III) chimeric channel.

Figure 2

CaMKIIγ_C forms a signaling complex with α_1H channels dependent on the II-III loop. (A) Immunoblot of channel immunoprecipitates from HEK 293 cells transiently expressing FLAG-tagged α_1H wild-type or α_1H(GII-III) chimeric channels, most cotransfected with CaMKIIγ_C, using: goat IgG or anti-α_1H for the immunoprecipitation, anti-FLAG for verification of channel immunoprecipitation, and anti-CaMKII for detection of CaMKII coassociation. Input lysates show expression level of CaMKIIγ_C with 0.25 pmol purified CaMKIIγ_C standard for comparison. (B) An analysis of 9 experiments. Integrated optical band densities of FLAG and CaMKII immunoreactivities (IR) in α_1H(GII-III) channel immunoprecipitates expressed relative to immunoreactivities in wild type. (C) In vitro binding of purified CaMKIIγ_C (5 pmol/250 μl) to bead-bound channel intracellular domain–GST fusion proteins (2 pmol) of channel intracellular domains: N-terminus (N-term), I-II loop, II-III loop, III-IV loop, C-terminus. Shown are immunoblots of bound kinase detected by anti-CaMKII, with 0.5 pmol standard for comparison. Below is a GST blot for each fusion protein indicating nearly equal loading.

The identification of α_1H channels as a CaMKII-binding partner and the importance of Ser1198 for the modulation of channel gating classify α_1H channels as candidate CaMKII substrates. However, previously there was no direct evidence that Ser1198 was phosphorylated by CaMKII in intact cells. To evaluate Ser1198 phosphorylation in intact cells and tissue, we developed a novel phosphomotif-specific antibody (anti-RAEpS¹¹⁹⁸) and as a byproduct a motif-specific antibody (anti-RAES¹¹⁹⁸), enabling us to determine whether α_1H channels are authentic kinase substrates. The specificity of the affinity-purified antibody anti-RAEpS¹¹⁹⁸ was evaluated initially using recombinant II-III loop proteins that contained the wild-type sequence or an Ala point mutation at Ser1198. Anti-RAEpS¹¹⁹⁸ recognized only the wild-type II-III loop protein following CaMKII phosphorylation. This phosphorylation-dependent interaction was competed with excess phosphopeptide antigen (pS¹¹⁹⁸ peptide) but not with the non-phosphopeptide (S¹¹⁹⁸ peptide) (Figure 3) and contrasted with anti-RAES¹¹⁹⁸ that recognized the wild-type fusion protein without regard for its phosphorylation state. Moreover, mutation of Ser1198 to Ala precluded recognition by anti-RAEpS¹¹⁹⁸ and anti-RAES¹¹⁹⁸. Thus, anti-RAEpS¹¹⁹⁸ exhibits appropriate phosphorylation-dependent specificity in vitro.

Figure 3

Characterization of phosphomotif-specific antibody targeting RAEpS¹¹⁹⁸ . Immunoblots of in vitro phosphorylation reactions of GST–α_1H II-III loop fusion proteins (1 μM) — wild-type (S¹¹⁹⁸) and mutant (A¹¹⁹⁸) — catalyzed by 10 nM CaMKIIγ_C (Ca²⁺ [0.5 mM], CaM [2 μM] ± ATP [100 μM]) showing selective recognition of pS¹¹⁹⁸ by anti-RAEpS¹¹⁹⁸ (top row, lanes 1, 5, 7, 9). Below is an anti-GST immunoblot for each fusion, indicating equal loading (middle row, lanes 1–4). Notably, anti-RAES¹¹⁹⁸ recognizes S¹¹⁹⁸ without regard for phosphorylation state (bottom row, lanes 1 and 2). Anti-RAEpS¹¹⁹⁸ preadsorption with phosphopeptide (Peptide-pS, lanes 11–14) but not non-phosphopeptide (Peptide, lanes 7–10) suppressed anti-RAEpS¹¹⁹⁸ immunoreactivity, showing phosphorylation-dependent interaction, confirming phosphomotif specificity.

We generated a HEK 293 cell line stably expressing a mouse leak K⁺ channel (mTREK) and FLAG-tagged α_1H Ca²⁺ channel (α_1H/mTREK) for use in intact cell phosphorylation studies. Ca²⁺ channel currents are robust in these cells, and 6 mM K⁺ depolarizes the cell an approximate 20 mV, as indicated in Figure 4A by the rightward shift in the zero current potential of the macroscopic currents, which were measured using a voltage-ramp protocol. Six millimolar K⁺ depolarized the membrane potential from –83 ± 4 mV at 2 mM K⁺ to –65 ± 3 mV (n = 5). Six millimolar K⁺ depolarization also elicited a transient rise in intracellular Ca²⁺ concentration [Ca²⁺]_i, as measured by fluo-4 fluorescence (Figure 4B). This increase in [Ca²⁺]_i was fully dependent on extracellular Ca²⁺, dose-dependently attenuated by cellular preincubation with a blocker of LVA Ca²⁺ channel entry (mibefradil at 3 μM and 10 μM), and precluded in cells stably expressing mTREK alone, despite similar changes in membrane potential elicited upon cell depolarization with 6 mM K⁺ (–76 ± 3 mV at 2 mM K⁺, –52 mV ± 6 mV at 6 mM K⁺; n = 6). Taken together these data argue strongly that the measured rise in intracellular Ca²⁺ is mediated by α_1H channels.

Figure 4

CaMKIIγ_C -induced phosphorylation of α_1H channels in situ. (A) Sample currents from α_1H/mTREK, transiently expressing CaMKIIγ_C. Top: α_1H tail currents elicited upon repolarization to –60 mV (V_test = –50, –30, –10, 0, +10 mV). Bottom: Whole-cell currents elicited by a voltage ramp (–120 to –20 mV). Zero-current potential defines membrane potential (V_m); dashed line highlights V_m change. (B) [Ca²⁺]_i plotted in arbitrary units of fluo-4-fluorescence with time in α_1H/mTREK cells (filled circles, open squares, open triangles, filled triangles) or mTREK-only cells (open circles). Six millimolar K⁺ (6K) added at 16 seconds. The plain line indicates timed response to 2 mM K⁺ challenge in α_1H/mTREK cells. Traces are mean response of cells in 3 plate wells. Histograms plot peak (20 seconds) rise in [Ca²⁺]_i (mean ± SEM; n as indicated). Cells were preincubated with 100 μM Ca²⁺ or 10 μM mibefradil 30 minutes prior to 6 mM K⁺ challenge and during measurement. *P < 0.05, 2 mM K⁺ versus treatment (ANOVA, Student-Newman-Keuls method). (C) Immunoblot of channel-enriched immunoprecipitates (as obtained in Figure 2) from cells challenged with 2 (lanes 1 and 3) or 6 (lanes 2 and 4–8) mM K⁺ with (lanes 1–7) or without (lane 8) CaMKIIγ_C transfection. Channels were immunoprecipitated with a control IgG or anti-α_1H. Anti-FLAG verified immunoprecipitation, and anti-RAEpS¹¹⁹⁸ evaluated phosphorylation. Cells were preincubated with 100 μM Ca²⁺ or 10 μM mibefradil as in B (lanes 5–6). (D) Analysis of 8 immunoprecipitation experiments. Integrated optical band densities of FLAG and RAEpS¹¹⁹⁸ immunoreactivities in 6 mM K⁺–stimulated samples expressed relative to immunoreactivities in 2 mM K⁺ samples in CaMKII-transfected (filled circles) or nontransfected cells (open circles).

To investigate whether Ser1198 was phosphorylated in situ, we compared the state of phosphorylation of α_1H channels in CaMKIIγ_C-transfected α_1H/mTREK cells that had been incubated with 2 mM or 6 mM K⁺. To isolate the channel protein, we immunoprecipitated α_1H channels from whole-cell lysates using a goat α_1H-specific antibody directed against the C-terminal tail of the channel (anti-α_1H). Cell stimulation with 6 mM K⁺ increased [Ca²⁺]_i and also increased the phosphorylation state of the α_1H channel RAES¹¹⁹⁸ motif in cells expressing CaMKIIγ_C (Figure 4C). The detected increase in RAEpS site immunoreactivity was dependent on Ca²⁺ entry through α_1H channels, as it was appropriately attenuated by low calcium (100 μM) or mibefradil (10 μM) preincubation and, as expected, was not observed in the immunoprecipitate of goat IgG. To quantify the increase in phosphorylation state, we evaluated the fold increase in anti-RAEpS immunoreactivity (6 mM K⁺/2 mM K⁺) and compared it to anti-FLAG immunoreactivity in the immunoprecipitate using a mouse monoclonal FLAG antibody to detect the FLAG-tagged channels. In 8 experiments, K⁺-mediated depolarization increased α_1H channel RAEpS site immunoreactivity 2- to 4-fold in cells expressing CaMKIIγ_C (230% ± 40%; n = 8; P < 0.001) compared with FLAG immunoreactivity (Figure 4D), which remained unchanged (103% ± 0.03%; n = 8; NS). By contrast, α_1H channel RAEpS site immunoreactivity was not increased in the absence of CaMKII transfection (100% ± 0.04%; n = 5; NS). Thus, we conclude that Ser1198 on α_1H channels is a bone fide CaMKII phosphorylation site in situ and that this phosphorylation is controlled by membrane depolarization that activates the LVA channel.

Based on our in vitro CaMKII-binding experiments, we hypothesized that the phosphorylation of the RAES motif by CaMKII would disrupt the coassociation of CaMKII and the channel protein. To maximize the degree of kinase autoinhibition and promote docking on the channel, we immunoprecipitated the channel-kinase complex from α_1H/mTREK cells that were maintained at 2 mM K⁺ and transfected with CaMKIIγ_C. CaMKII in the immunoprecipitated complex was maintained in an inactive conformation or was activated with Ca²⁺/CaM plus ATP, either alone or following preincubation and maintained incubation with an active (10 μM KN-93) or inactive (10 μM KN-92) CaMKII inhibitor (Figure 5). In the absence of cellular activation, α_1H channels formed a complex with the kinase and showed no evidence of RAES site phosphorylation. Activation of the associated kinase in vitro promoted the phosphorylation of the RAES motif and the complete dissociation of the channel-kinase complex, which was prevented by preincubation with the active but not the inactive CaMKII inhibitor. Thus, autoinhibited CaMKII forms a stable complex with the channel that can be fully disrupted by CaMKII activity.

Figure 5

CaMKIIγ_C forms a signaling complex with α_1H channels that is disrupted by S¹¹⁹⁸ phosphorylation. Typical immunoblot of channel immunoprecipitates from FLAG- α_1H/mTREK double-stable cells incubated at 2 mM K⁺ expressing transfected CaMKIIγ_C, using a α_1H C terminus–specific antibody for immunoprecipitation and anti-FLAG for verification of channel immunoprecipitation. CaMKII in the immunoprecipitated complex was kept inactive or was activated with Ca²⁺/CaM + ATP, either alone or following preincubation and maintained incubation with 10 μM of a CaMKII inhibitor, KN-93, or its inactive analog, KN-92. Anti-CaMKII detected CaMKII retained in the immunoprecipitate, and anti-RAEpS¹¹⁹⁸ assessed the state of S¹¹⁹⁸ phosphorylation.

To confirm the specificity of the anti-RAEpS¹¹⁹⁸ antibody in the context of complex interacting cellular proteins, we evaluated the state of phosphorylation of the RAES motif in our α_1H/mTREK test system. As illustrated in representative immunoblots (Figure 6A), we detected strong anti-RAEpS¹¹⁹⁸ diaminobenzidine (DAB) immunostaining in response to 6 mM K⁺ depolarization. Notably, this staining was competed with the antigenic peptide (pS¹¹⁹⁸ peptide) but was not competed with the non-phosphopeptide (S¹¹⁹⁸ peptide). By contrast anti-RAEpS¹¹⁹⁸ DAB immunostaining of cells maintained at 2 mM K⁺ was weak. Thus, we conclude that anti-RAEpS¹¹⁹⁸ immunoreactivity reports the phosphorylation state of the RAES motif and immunohistochemistry can be used to determine whether Ser1198 phosphorylation of native LVA channels is also modulated in vivo.

Figure 6

Phosphorylation-state of Ser1198 in α_1H channels in situ and in vivo. Immunohistochemical detection of pS¹¹⁹⁸ in: CaMKIIγ_C-transfected α_1H/mTREK double-stable cells following 6 mM K⁺ depolarization (1 minute) (A); thin sections of rat adrenal glands harvested 30 minutes after vehicle or Ang II infusion, 50–200 ng/kg/min (n = 4 and 7 animals, respectively) (B); thin sections of rat adrenal glands subcapsularly perfused (1 μl/min) with D5W with or without 100 μM KN-93 30 minutes before and during a 30-minute systemic infusion of Ang II at 50 ng/kg/min in uniadrenalectomized rats (n = 3 animals each) (C). Anti-RAEpS¹¹⁹⁸ immunohistochemistry revealed DAB immunostaining in the subcapsular ZG (B and C). Note the increase in signal strength after stimulation (A–C). Signal competed with an 80-fold molar excess of antigenic phosphopeptide, pS¹¹⁹⁸ peptide, but not non-phosphopeptide, S¹¹⁹⁸ peptide, evaluated at either ×40 (B, bottom row; and C) or ×100 (B, top row; and A). All samples were counterstained with hematoxylin. Quantification of ZG DAB immunostaining expressed as percent of ROI. ^#P < 0.05, control S¹¹⁹⁸ peptide versus antibody alone. ^##P < 0.05, stimulated pS¹¹⁹⁸ peptide versus antibody alone (ANOVA on ranks, Student-Newman-Keuls method). ^†P < 0.05, KN-93 versus vehicle, infused using the primary antibody alone or with pS¹¹⁹⁸ peptide preadsorption (ANOVA, Student-Newman-Keuls method).

Ang II is a major physiological regulator of aldosterone production from cells of the ZG that robustly express α_1H channels (38, 39). Moreover, Ang II activates CaMKII (40), and CaMKII activity is important for sustaining Ang II–stimulated aldosterone secretion (41, 42). Therefore, we harvested adrenal glands from rats following a 30-minute infusion of Ang II (50–200 ng/kg/min) or saline vehicle and compared the phosphorylation of Ser1198 by immunohistochemistry (Figure 6B) to localize the α_1H antigen within the ZG and to avoid its dilution with adrenal proteins from other regions. To blunt the fright response and the attendant surge in release of ACTH, itself an aldosterone secretagogue, we administered dexamethasone to the animals 24 hours before study (see Methods). Ang II infusion raised plasma aldosterone concentration 4-fold, from 247 ± 105 to 978 ± 92 pg/ml (mean ± SEM; n = 4 and 7 animals, respectively; P < 0.004). Following Ang II stimulation, we detected strong anti-RAEpS¹¹⁹⁸ DAB immunostaining among cells of the ZG that reside directly underneath the capsular envelope (Figure 6B, Ang II/primary Ab). Significantly, this tissue staining was competed with the antigenic pS¹¹⁹⁸ peptide (Figure 6B, Ang II + pS¹¹⁹⁸ peptide) but not with the S¹¹⁹⁸ peptide (Figure 6B, Ang II + S¹¹⁹⁸ peptide) and replicated the zonal distribution of α_1H mRNA detected by in situ hybridization (39). Sections from control tissue (Figure 6B, Control) exhibited consistently weaker DAB immunostaining than those from stimulated tissue (Figure 6B, Ang II). Quantification of anti-RAEpS¹¹⁹⁸ DAB immunostaining of the ZG (Figure 6C) revealed a 6-fold increase in the staining of Ang II–stimulated tissue (3.9% ± 0.7% to 24.7% ± 3.9% region of interest [ROI]; P < 0.006) that was reduced by approximately 60% by preadsorption with an 80-fold molar excess of antigenic phosphopeptide, establishing the α_1H channel as a bona fide kinase substrate in vivo.

To determine whether CaMKII was indeed mediating the Ang II–elicited change in S¹¹⁹⁸ phosphorylation in vivo, we endeavored to blunt CaMKII activation by direct delivery of the water-soluble, cell-permeant CaMKII inhibitor (KN-93) to the subcapsular interstitium of the adrenal cortex. Following uniadrenalectomy and a short surgical recovery period, the remnant adrenal gland was infused in vivo (1 μl/min, 30 minutes) with 5% dextrose (D5W) or D5W containing 100 μM KN-93, prior to and during a systemic infusion of Ang II (50 ng/kg/min, 30 minutes). Blood samples were taken for aldosterone measurement after uniadrenalectomy before the onset and upon the termination of subcapsular infusion to determine the secretory response of the remnant adrenal to Ang II infusion. The remnant gland was harvested, and the phosphorylation state of Ser1198 was compared with that of its preinfused paired control (Figure 6C). As illustrated in Figure 6C and quantified in the top panel, the direct cortical delivery of KN-93 abrogated the 2-fold increase in RAEpS¹¹⁹⁸ immunostaining of the Ang II–stimulated tissue (20% ± 5.7% ROI [Ang II] versus 3.3% ± 2.7% ROI [Ang II + KN-93]); n = 3 each; P < 0.05) and concomitantly blunted the aldosterone secretory response of the remnant adrenal to Ang II (1,004 ± 179 pg/ml [Ang II]; 569 ± 40 pg/ml [Ang II + KN-93]); n = 3 each; P < 0.05). Taken together, our data indicate the participation of a Ca²⁺/CaM-dependent kinase in the dynamic regulation of Ser1198 by physiological stimuli that activate aldosterone secretion and suggest an important role for channel regulation in the control of aldosterone secretion.

Despite decades of research on the regulation of voltage-gated ion channels by protein phosphorylation, few phosphorylation events that alter ion channel activity have been shown to be physiologically modulated in vivo (43–45). This is especially true of the positive feedback regulation of L-type and T-type channels by CaMKII. CaMKII-induced facilitation of L-type Ca²⁺ channels requires active kinase (16, 46, 47), yet the phosphorylation site(s) of functional importance have eluded identification (21, 48).

Previous studies suggested that Ser1198 in α_1H channels is required for the potentiation of T-type Ca²⁺ channel current by CaMKII, implicating protein phosphorylation as the mechanism by which CaMKII regulates α_1H channels (23). In addition, single-channel experiments using membrane patches excised from native bovine ZG cells demonstrated that CaMKII is contained within the domain of the excised patch, suggesting that channel regulatory elements are locally contained (19). The present study significantly enhances our understanding of the nature of CaMKII–α_1H channel interactions and the existence of a CaMKII-channel signaling complex. Our findings provide a biochemical and molecular explanation for how CaMKII modulates LVA Ca²⁺ channels and show that a physiological agonist activates this mechanism to enhance the phosphorylation of α_1H channels at Ser1198 in vivo.

We have uncovered what we believe to be a novel mechanism for Ca²⁺-dependent autoregulation of α_1H channels that exploits the changing conformational states of CaMKII. A cycle of autoactivation begins with the docking of inactive CaMKII on the II-III loop of α_1H channels under basal conditions (Figure 7A). As a binding partner of the II-III loop, CaMKII is well positioned to sense local Ca²⁺ concentrations that arise from the opening of the LVA channel itself (Figure 7B). The reception of this signal by the kinase induces its activation as well as the transfer of signaling information to Ser1198 as CaMKII shifts its primary interaction with the II-III loop to its catalytic domain as it initiates phosphotransfer (Figure 7C). As is characteristic of interactions between a protein kinase and its substrates (49), phosphorylation of Ser1198 disrupts this association (Figure 7D), dissociating the kinase either to begin a new cycle of autoregulation at another channel or to execute other functions.

Figure 7

Four-stage mechanistic model of CaMKII and α_1H channel interactions. (A) Inactive CaMKII docks on the II-III loop of α_1H Ca²⁺ channels poised to sense Ca²⁺ influx. (B) Depolarization-mediated Ca²⁺ influx elicits a local rise in Ca²⁺ and activates CaMKII. (C) Activated CaMKII migrates to S1198 for phosphotransfer. (D) CaMKII phosphorylates S¹¹⁹⁸, provoking further Ca²⁺ influx, and then dissociates. Membrane potential is restored, and the cycle begins anew.

These interaction features describe a new paradigm for modulation of ion channel substrates by CaMKII. Inactive kinase does not significantly interact with the cytoplasmic regions of NMDA receptors (31–35), ether-a-go-go channels (36, 37), ryanodine receptors (50), or α_1C L-type Ca²⁺ channels (21). Moreover, the recruitment of active kinase to these channels forms long-lasting associations when the kinase is autophosphorylated and new sites of interaction are exposed (T sites). In most instances T site binding produces constitutive kinase activity, keeping the kinase tethered in a catalytically active conformation in the absence of Ca²⁺ elevation or autophosphorylation (31–37).

Notably, our data show that the proposed mechanism for α_1H channel modulation by CaMKII is engaged in a model cell by changes in membrane potential that are induced by extracellular K⁺ at concentrations that physiologically stimulate ZG cells. Elevation in extracellular K⁺ from 2 to 5 mM potentiates the aldosterone secretory response to physiological doses of Ang II (51, 52). Thus, although extracellular K⁺ and Ang II can be considered independent regulators of aldosterone production, the physiological potency of these agonists depends on their combined activities and their synergy. Our data provide strong evidence for the operation of this mechanism in the adrenal ZG in response to Ang II signaling in vivo. Thus, Ser1198 in α_1H channels is, to our knowledge, the first residue in voltage-gated T-type Ca²⁺ channels shown to be dynamically phosphorylated in vivo. The targeted disruption of docking and/or CaMKII-mediated phosphorylation of α_1H channels may offer an alternative therapeutic strategy to that of mineralocorticoid receptor antagonism for limiting the development and progression of cardiovascular damage in heart failure.

We acknowledge support from NIH grants (HL36977 to P.Q. Barrett and MH63232 to R.J. Colbran) and American Heart Association MidAtlantic-Affiliate postdoctoral fellowships to J. Yao and L.A. Davies.

Address correspondence to: Paula Q. Barrett, Department of Pharmacology, University of Virginia School of Medicine, 1300 Jefferson Park Avenue, Charlottesville, Virginia 22908, USA. Phone: (434) 924-5454; Fax: (434) 982-3878; E-mail: pqb4b@virginia.edu .

Nonstandard abbreviations used: [Ca²⁺]_i, intracellular [Ca²⁺]; CaM, calmodulin; CaMKII, Ca²⁺/calmodulin-dependent protein kinase II; D5W, 5% dextrose; GST, glutathione-S-transferase; HVA, high voltage–activated; LVA, low voltage–activated; PFA, paraformaldehyde; ROI, region of interest; ZG, zona glomerulosa.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.116:2403–2412 (2006). doi:10.1172/JCI27918.

J.D. Howard’s present address is: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

P.J. Welsby’s present address is: Department of Physiology, Trinity College, Dublin, Ireland.

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