Isolation of Arterial and Adult and Neonatal Cardiac Myocytes

Mice and rats were euthanized as approved by the University of Washington Institutional Animal Care and Use Committee. Adult arterial myocytes and neonatal cardiac myocytes were prepared as described previously.^5,10

Cav1.2 and Calmodulin Constructs and Inducible PKCα Translocation System and Their Expression in tsA-201 Cells

We transfected tsA-201 cells using JetPEI (Polyplus Transfection) with plasmids encoding calcium channel pore-forming and accessory subunits as well as CaM. We generated the rabbit homolog of the human TS Cav1.2 (G436R, rabbit; G406R human).¹¹ Wild-type (WT) CaM and the Cav1.2 mutant with a stop codon at amino acid 1670 (Cav1.2Δ1670X) were kindly provided by Dr David T. Yue (Departments of Biomedical Engineering and Neuroscience, The Johns Hopkins University, Baltimore, Md). We generated a rapamycin-induced PKCα translocation system similar to the one developed by Liberles et al¹² and Suh et al.¹³ Reagents for this system were obtained from Agilent Technologies and Dr John Exton (Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tenn) (PKCα).

Coupled Markov Chain Model

We implemented a coupled Markov chain model in Matlab to determine the coupling coefficient (κ) among Cav1.2 channels underlying membrane currents and Ca²⁺ sparklet sites. The model was originally developed by Chung and Kennedy¹⁴ to analyze individual records of partially coupled GABA-activated chloride channels.

Electrophysiology, Confocal, and Total Internal Reflection Fluorescence Microscopy

Voltage-clamp experiments were performed using standard patch-clamp techniques. Total internal reflection fluorescence (TIRF) images were acquired using a through-the-lens TIRF microscope. Images were acquired at 100 to 200 Hz. Ca²⁺ sparklets were detected and defined for analysis using an automated algorithm.⁵ Some experiments involved a Nikon swept field confocal system.

Statistics

Data are presented as means±SEM. A probability value of less than 0.05 was considered significant. The asterisk (*) symbol is used in the figure to illustrate a significant difference between groups.

Optical Recordings of Coupled L-Type Ca²⁺ Channels

A TIRF microscope was used to image Ca²⁺ sparklets in arterial smooth muscle cells, neonatal ventricular myocytes, and tsA-201 cells expressing WT Cav1.2 channels. All Ca²⁺ sparklet experiments were performed in voltage clamped cells using the whole-cell configuration of the patch-clamp technique. Ca²⁺ sparklets were recorded in cells treated with the SERCA pump inhibitor thapsigargin (1 μmol/L) to eliminate Ca²⁺ release from intracellular Ca²⁺ stores. To image Ca²⁺ sparklets, cells were dialyzed with a patch pipette solution containing the fluorescent Ca²⁺ indicator fluo-5F and EGTA. The inclusion of the relatively slow Ca²⁺ buffer EGTA (on rate ≈100-fold slower than fluo-5F) in the intracellular solution serves an important purpose: it restricts fluo-5F fluorescence to the site of Ca²⁺ entry (≈1 μm). This occurs because with EGTA in the cytosol, Ca²⁺ entering the cell initially interacts with the faster fluo-5F, producing a fluorescence signal, but then quickly (≈2 ms) binds to the more abundant and nonfluorescent EGTA. Thus, in our TIRF experiments, [Ca²⁺]_i signals are limited to the submembrane space near the mouth of surface membrane Ca²⁺ channels.

Figure 1A shows representative Ca²⁺ sparklets from arterial smooth muscle cells, neonatal ventricular myocytes, and tsA-201 cells expressing Cav1.2 channels. These Ca²⁺ sparklets were recorded while cells were held at −70 mV to increase the driving force for Ca²⁺ influx and maintain a low L-Type Ca²⁺ channel activity, which increased contrast and hence our ability to detect discrete Ca²⁺ entry sites. A Δ[Ca²⁺]_i amplitude histogram of these records revealed 2 discrete peaks corresponding to closed channels (Δ[Ca²⁺]_i=0 nmol/L) and openings of ≈38 nmol/L, which as demonstrated previously, represents the amplitude of quantal Ca²⁺ sparklet events in tsA-201 cells expressing Cav1.2 and arterial myocytes.^5,6,15 A previous study⁶ suggested that quantal Ca²⁺ sparklets are likely produced by the opening of a single Ca²⁺ channel.

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Figure 1. Optical recordings of coupled gating of Cav1.2 channels. A, Representative records of Ca²⁺ influx via Cav1.2 channels in neonatal cardiac myocytes (top), tsA-201 cells (middle), and arterial myocytes (bottom). B, All-points histogram of Ca²⁺ sparklet traces in A. The solid black line is the fit (χ²=2.1) to the data with a multicomponent Gaussian function with centers at 0 and 38 nmol/L. C, Δ[Ca²⁺]_i from a Ca²⁺ sparklet site in an arterial myocyte with coupled Cav1.2 channels. D, All-points histogram of the Ca²⁺ sparklet trace in C. The solid black line is the best-fit (χ²=2.5) to the data with a 3-component Gaussian function with centers at 0, 120, and 160 nmol/L. E, Histogram of the coupling coefficients (κ) of multiple Ca²⁺ sparklet records from cardiac, arterial, and tsA-201 cells. F, Ca²⁺ sparklet records with varied κ values.

It is important to note that, consistent with previous work,^5,6 Ca²⁺ sparklet activity in smooth muscle cells, neonatal ventricular myocytes, and tsA-201 cells transfected with Cav1.2 was rapidly eliminated by the application of the L-Type Ca²⁺ channel inhibitor nifedipine (10 μmol/L) or by superfusion of a Ca²⁺-free external solution (data not shown). Importantly, and as reported recently,⁵ Ca²⁺ sparklets were never observed in untransfected tsA-201 cells. Furthermore, Ca²⁺ sparklets in arterial and neonatal ventricular myocytes have the same amplitude of quantal event, activity, and pharmacology (eg, insensitive to 1 μmol/L thapsigargin, eliminated by 10 μmol/L nifedipine) as Ca²⁺ sparklets in tsA-201 cells expressing Cav1.2 channels.^5,15 Collectively these observations strongly suggest that Ca²⁺ sparklets in arterial smooth muscle cells, neonatal ventricular myocytes, and tsA-201 cells expressing Cav1.2 channels are produced by Ca²⁺ influx via L-Type Ca²⁺ channels.

Although most Ca²⁺ sparklets recorded in these cells were similar to those in Figure 1A, we noticed that in a number of Ca²⁺ sparklet sites (191 of 491 sites) small clusters of Cav1.2 channels opened and closed simultaneously to promote greater elevation in subcellular [Ca²⁺]_i (Figure 1C). The Δ[Ca²⁺]_i histogram of the trace in Figure 1D had 3 distinct peaks at a Δ[Ca²⁺]_i of 0, 120, and 160 nmol/L. The peaks at 120 and 160 nmol/L likely resulted from the simultaneous activation of 3 and 4 Cav1.2 channels, respectively. Note the absence of a peak near the quantal amplitude of Ca²⁺ influx (ie, 38 nmol/L) in this record. Examples of records with sequential quantal and multi-channel openings are shown in Figures 4A and 5⇓A below.

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Figure 4. Displacing CaM from the IQ domain of Cav1.2 increases coupled gating of WT and TS Cav1.2 channels. A, Ca²⁺ sparklet records from tsA-201 and arterial myocytes showing induction of coupled gating by W7 (100 μmol/L). Kappa values are shown above each trace. B, Number of coupled sites per cell before and after W7. C, Illustration of Cav1.2-EGFP and CaM-tRFP. Right, Images of Cav1.2-EGFP in tsA-201 cells under control conditions, during W7 treatment, and following CaM-tRFP photobleaching. D, Cav1.2-EGFP fluorescence intensity at an example cross-section of the membrane marked by the white line in each of the images in C. Dotted lines show the F_DA and F_A values used to calculate effective FRET=1−(F_DA/F_A). E, FRET in specific sites before and after W7 (center, lower row) or PDBu (right, lower row) application. Yellow bars are mean values. F, Ca²⁺ sparklet records with coupled Cav1.2 channels from tsA-201 cells expressing Cav1.2-TS and hypertensive smooth muscle. G, Representative Cav1.2 channel currents evoked by depolarization from −80 to −30 mV in patches (cell-attached) from tsA-201 cells expressing WT (upper trace) and Cav1.2-TS channels (lower trace). The close state is denoted by a “c” and the open state by an “o.” H, Number of sites with coupled Cav1.2 channels per cell in cells expressing WT and TS Cav1.2 and in normotensive (NT) and hypertensive (HTN) smooth muscle. I, FRET data between EGFP-tagged WT or TS Cav1.2 and CaM-tRFP.

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Figure 5. AKAP150, but not PKCα, is required for coupled gating of Cav1.2 channels. A, Representative Δ[Ca²⁺]_i records from Ca²⁺ sparklet sites in AKAP150^−/− and PKCα^−/− smooth muscle cells under control conditions (right) and after exposure to 100 μmol/L W7. The coupling coefficient (κ) of these Cav1.2 channels is shown above each trace. Dotted lines show the amplitude of quantal levels. B, Bar plot of the mean number of coupled sites per cell.

The observation of Ca²⁺ sparklets resulting from the simultaneous openings of small clusters of Cav1.2 channels is surprising because the P_o of a single Cav1.2 channel is only ≈10⁻⁶ at −70 mV.¹⁶ The probability of independently gating channels opening simultaneously at this potential should be very low: ≈10⁻¹², 10⁻¹⁸, and 10⁻²⁴ for the random, coincident opening of 2, 3, or 4 channels. Thus, the probability of observing simultaneous openings of Ca²⁺ channels should be progressively lower as the number of participating channels increase, which should result in a histogram with peaks of decreasing amplitudes as multichannel events become rarer. On the basis of that analysis, Ca²⁺ sparklet events like the one in Figure 1C should be very rare. Yet, Ca²⁺ sparklets resulting from the coincident activation of 2 or more Cav1.2 channels were frequently observed. This suggests that this is not simply the consequence of overlapping stochastic openings of independently activated Cav1.2 channels. Rather, our data provide evidence for the coordinated activation of a small cluster of these channels.

To test this hypothesis, we implemented a coupled Markov chain model to determine the coupling coefficient (κ) among Cav1.2 channels within Ca²⁺ sparklet sites (Figure 1E and 1F). The model was originally developed by Chung and Kennedy¹⁴ and used to analyze individual records of partially coupled GABA-activated chloride channels. Briefly, this model is a simplified Markov chain model with only 3 free parameters that takes the form of a set of binary chains that are interdependent according to a lumped coupling coefficient parameter called κ. The other 2 parameters (called ρ and ς) describe the intrinsic characteristics of the binary chains. The κ value could range from 0 for independently gating channels to 1 for “tightly” coupled channels that open and close simultaneously all the time. A detailed description of this model is provided in the expanded Methods section (Online Data Supplement).

Using this analysis, we found that although the majority of Ca²⁺ sparklets (59%) were likely the result of Ca²⁺ influx via independently gating Cav1.2 channels (ie, κ=0), numerous Ca²⁺ sparklets were produced by Cav1.2 channels with a κ > 0. Indeed, the coupling coefficient of nonindependent events (ie, κ > 0) ranged from 0.1 to 1 with a median value of 0.2. Together with the Ca²⁺ sparklet data above, this Markov analysis suggested that a subpopulation of Cav1.2 channels was capable of gating coordinately.

Electrophysiological Recordings of Coupled L-Type Ca²⁺ Channels

We used electrophysiological techniques to record L-Type Ca²⁺ channel currents (Figure 2). In these experiments, we recorded elementary L-Type Ca²⁺ channel currents from cell-attached patches from neonatal ventricular myocytes. L-Type Ca²⁺ channel currents were evoked by a 2 s step depolarization to −30 mV from the holding potential of −80 mV. The patch pipette solution contained 20 mmol/L Ca²⁺ (charge carrier), 10 μmol/L tetrodotoxin, 130 mmol/L TEA⁺ and no Na⁺ or K⁺ to eliminate Na⁺ and K⁺ channel currents. The L-Type Ca²⁺ channel opener BayK-8644 (500 nmol/L) was included in the pipette solution to increase the mean open time and P_o of these channels and the probability of PKCα-dependent, persistent L-Type Ca²⁺ channel activity.⁵

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Figure 2. Electric recordings of coupled L-Type Ca²⁺ channels. A, Representative elementary L-Type Ca²⁺ channel records obtained during a step depolarization (2 seconds) from −80 to −30 mV. Openings are shown as downward deflections, with the close state denoted by “c,” and the open state by “o.” B, Histogram of L-Type Ca²⁺ currents traces in A. The solid black line is the fit to the data with a multicomponent Gaussian function with centers at 0.0 and 0.5 pA. The inset shows a histogram from a representative record obtained in the presence of 10 μmol/L nifedipine. C, Representative single and coupled L-Type Ca²⁺ channel current records obtained during step depolarizations from −80 to −30 mV. D, Histogram of the L-Type Ca²⁺ currents records in C. The solid black line is the fit to the data with a multicomponent Gaussian function with centers at 0.0, −0.5, −1.0, and −2.9 pA. E, Trace shows a portion (gray box) of the record in C showing a L-Type Ca²⁺ current resulting from the simultaneous opening and closing of multiple channels. A histogram from this portion of the trace is shown below. The histogram was fitted with a Gaussian function with centers at 0.0 and −3.1 pA. F, Histogram of the coupling coefficients (κ) of multiple L-Type Ca²⁺ current records from cell-attached patches from neonatal ventricular myocytes.

As expected from our Ca²⁺ sparklet studies, depolarization to −30 mV evoked relatively low levels of Ca²⁺ channel activity in most (112 of 134 sweeps, or 84%; from 5 cells) of the sweeps examined (Figure 2A). A current amplitude histogram from these records show 2 distinct peaks with centers at 0 pA (ie, closed channel) and 0.53 pA, which corresponds to the expected single channel current level for an L-Type Ca²⁺ channel at −30 mV with 20 mmol/L Ca²⁺¹⁶. The κ value of the Cav1.2 channels in these patches was 0, suggesting that these currents were produced by independently gating channels. At this point it is important to note that in control experiments inclusion of 10 μmol/L nifedipine prevented all Ca²⁺ channel activity, indicating that L-Type Ca²⁺ channels produced these currents (inset in Figure 2B).

Consistent with our Ca²⁺ sparklet data, we found that in a minority of the records (22 out 134 sweeps or 16%; from 5 cells) membrane depolarization to −30 mV evoked elementary as well as relatively large currents that were likely produced by the simultaneous opening and closing of multiple L-Type Ca²⁺ channels (Figure 2C). The amplitude histogram of the current records in Figure 2C had 4 prominent peaks at 0.0, 0.5, 1.0, and 2.9 pA, likely resulting from the activation of a single or simultaneous activation of 2 or 6 L-Type Ca²⁺ channels, respectively. Closer examination (Figure 2E) of one of the large current events highlighted by the gray box in the bottom sweep in Figure 2C, indicates that in this patch 5 to 6 channel likely opened and then closed simultaneously multiple times. Three additional multichannel current events of similar amplitude were observed in this sweep. As noted above, the prominence of the peak with a center at 3.1 pA in the histogram in Figure 2E and absence of peaks of larger amplitude between this peak and the peak at 0 pA suggests coupled gating between L-Type Ca²⁺ channels. Consistent with this, the coupling coefficient of the L-Type Ca²⁺ channels in this section was 0.22, suggesting this current was produced by partially coupled channels. Indeed, analysis of all L-Type Ca²⁺ channel records indicates that the majority of the currents were likely the result of independent openings of L-Type Ca²⁺ channels (ie, κ=0; Figure 2F). In combination with the Ca²⁺ sparklet data above, these findings strongly support the hypothesis that small clusters of Cav1.2 channels can open and close coordinately.

Activation of PKCα Increases Coupled Gating of Cav1.2 Channels

Activation of PKCα has been linked to elevated persistent Ca²⁺ sparklet activity in arterial myocytes.^5,6,9 Thus, we investigated whether membrane translocation and activation of this kinase increases coupled gating of Cav1.2 channels. To do this, we used 2 complementary approaches. First, we generated a chemically-induced PKCα translocation system.^12,13 Briefly, we created an adenoviral vector expressing a FKBP12-rapamycin-binding element (FRB) that is anchored to the plasma membrane via the myristoylation and palmitoylation modification sequence Lyn₁₁. This adenoviral vector also expressed PKCα fused to the enhanced green fluorescent protein (EGFP) and a FK506 binding protein. Application of rapamycin promoted heterodimerization of protein domains from FKBP-PKCα-EGFP and the membrane anchored FRB. This recruited FKBP-PKCα-EGFP to the membrane from the cytosol on addition of rapamycin (Figure 3A). We used this inducible PKCα in neonatal ventricular myocytes and tsA-201 cells expressing Cav1.2. We found that induction of membrane translocation of PKCα (Figure 3A) increased Ca²⁺ sparklet activity and coupled gating of Cav1.2 channels in cardiac myocytes (n=5 cells; Figure 3B) and tsA-201 cells (n=6 cells; Online Figure I).

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Figure 3. Activation of PKCα increases coupled gating between Cav1.2 channels. A, Confocal images of neonatal ventricular cells expressing the inducible PKCα translocation system before (0 second) and after (75 and 150 seconds) application of 0.1 μmol/L rapamycin to induce membrane translocation of this kinase. The graph on the right shows the time course of PKCα fluorescence intensity in the membrane and cytosol in this cardiac myocyte. Δ[Ca²⁺]_i records from multiple Ca²⁺ sparklet sites before (left) and after (right) induction of PKCα-EGFP translocation are shown below the confocal images (B). The κ of the Cav1.2 channels in this site is shown above each record. C, Ca²⁺ sparklet records from an arterial myocyte before and after PDBu (500 nmol/L). D, Bar plot of the mean number of coupled sites per cell in arterial myocytes under control condition and after application of 500 nmol/L PDBu. E, Relationship between Ca²⁺ sparklet activity and coupling coefficient. Red line is a linear fit to the data (r²=0.64).

Our second approach involved the activation of endogenous PKC using the phorbol 12,13-dibutyrate (PDBu) (500 nmol/L). Activation of PKC with PDBu had a similar effect in arterial myocytes (n=17 cells; Figure 3C and 3D). Taken together, these data suggest that membrane translocation and activation of PKCα increases the probability of coupled gating between Cav1.2 channels.

Movement of Calmodulin Away From the IQ Domain in the C-Tail of Cav1.2 Channels Increases Coupled Gating of These Channels

We observed that the activity of Ca²⁺ sparklets produced by coupled Cav1.2 channels was higher than those produced by independently gating channels (Figure 3E). This raised the intriguing possibility that the probability of coupled gating of Cav1.2 channels might be enhanced by deficient Ca²⁺-dependent inactivation (CDI) of these channels. CaM mediates CDI of Cav1.2 channels.^17,18 Thus, we tested the hypothesis that inhibition of CaM increases the probability of coupled gating of Cav1.2 channels.

Consistent with this hypothesis, we found that application of the CaM antagonist W7 (100 μmol/L) increased the number of sites with coupled Cav1.2 channels ≈7-fold and 27-fold in tsA-201 cells (n=4 cells) and myocytes (n=7 cells), respectively (P<0.05; Figure 4A and 4B). Similar findings were obtained in myocytes injected with the CaM-inhibitory peptide (1 μmol/L; n=4 cells; expanded Methods section [Online Data Supplement] and Online Figure II). Application of either CaM antagonist increased the number of Ca²⁺ sparklet sites with coupled Cav1.2 channels, but not the median κ value (0.2) of coupled channels (P>0.05).

To examine the dynamics of this phenomenon, we used FRET to visualize the interaction between Cav1.2 channels and CaM. The C terminus of Cav1.2 was tagged with the EGFP (Cav1.2-EGFP) and CaM was fused with tag red fluorescent protein (CaM-tRFP) (Figure 4C and 4D). CaM associates with the IQ domain in the C terminus of Cav1.2, which is critical for CDI of these channels.^17,19 Accordingly, FRET was relatively high (0.32±0.02, n=10 cells) between Cav1.2-EGFP and CaM-tRFP under control conditions. Application of the CaM antagonist W7 (100 μmol/L) or the PKC activator PDBu (500 nmol/L) decreased FRET between Cav1.2-EGFP and CaM-tRFP from 0.21±0.04 to 0.08±0.03 (n=10 cells) and from 0.33±0.02 to 0.12±0.10 (n=11 cells), respectively (Figure 4E) (P<0.05). Note, however, that the magnitude of the decrease in FRET induced by W7 and PKCα activation varied within cells. The relative change in FRET ranged from 0 in some sites (red line in Figure 4E) to 100% in others (green line in Figure 4E). A decrease in FRET suggests an increase in the distance between Cav1.2-EGFP and CaM-tRFP. This could result from 2 potential scenarios. First, CaM-tRFP dissociates from the IQ domain of Cav1.2-EGFP channels. Second, CaM-tRFP changes in position within the C tail of Cav1.2 channels, moving away from the EGFP fused to the C terminus of these channel enough to decrease or even eliminate FRET. Although our FRET experiments cannot distinguish between these 2 possibilities, they evidently suggest that displacement of CaM away from the IQ domain of Cav1.2 channels may be a critical step in the induction of coupled gating of these channels.

Increased Coupled Gating of Cav1.2 Channels During Hypertension and TS

Increased Cav1.2 channel activity underlies lethal cardiac arrhythmias and autism in humans with TS.²⁰ In TS, a single amino acid substitution (Gly-436-Arg) in Cav1.2 (Cav1.2-TS) increases the open time and P_o of these channels.^11,20 We recorded Ca²⁺ sparklets (−70 mV) and depolarization-evoked currents in tsA-201 cells expressing WT and Cav1.2-TS channels (Figure 4F and 4G, respectively). We found a higher frequency of Ca²⁺ sparklets with coupled Cav1.2 channels in tsA-201 cells expressing Cav1.2-TS (n=10 cells) than WT channels (n=10 cells; Figure 4H; P<0.05).

Next, we used experimental conditions similar to those used to record Ca²⁺ currents in neonatal cardiac myocytes above. Membrane depolarization to −30 mV activated WT and Cav1.2-TS channels in tsA-201 cells. Voltage-activated Ca²⁺ currents were never recorded in untransfected tsA-201 cells. As in neonatal myocytes, the amplitude of the elementary Ca²⁺ currents activated by a voltage pulse to −30 mV was similar (0.49±0.01 pA) to that in neonatal ventricular myocytes (0.53±0.01 pA; P>0.05). This observation gives further support to the view that Cav1.2 channels produced the currents recorded in neonatal myocytes. Consistent with our Ca²⁺ sparklet studies, we recorded a number of Ca²⁺ currents resulting from the simultaneous opening and closing (ie, coupled gating) of channels in cells expressing Cav1.2-TS than WT Cav1.2 (Figure 4G).

Increased L-Type Ca²⁺ channel activity in arterial myocytes has been implicated in the chain of events that leads to vascular dysfunction during hypertension.^21,22 In hypertensive arterial myocytes, higher Cav1.2 channel activity is due, at least in part, to increased local PKCα activity. We found a higher frequency of coupled Cav1.2 channel gating events both in arterial myocytes from an animal model of angiotensin II–induced hypertension and in tsA-201 cells expressing Cav1.2-TS channels (Figure 4F and 4H).

We investigated whether, as with WT Cav1.2 channels, the higher frequency of coupled gating events of Cav1.2-TS channels was associated with displacement of CaM from the IQ domain in the C terminal of these channels. Accordingly, FRET between Cav1.2-TS-EGFP and CaM-tRFP was undetectable (n=13 cells; Figure 4I). These data suggest that coupled gating of Cav1.2 channels is associated with displacement of CaM away from the IQ domain in the C-terminal tail and contributes to enhanced channel function and Ca²⁺ influx during hypertension and TS.

AKAP150 Increases the Probability of Coupled Gating Between Cav1.2 Channels

The scaffolding protein AKAP150 targets PKCα to the cell membrane²³ and interacts with the C tail of Cav1.2 channels.²⁴ Therefore, we tested the hypothesis that PKCα activation and its targeting to the membrane by AKAP150 is necessary to induce coupled gating of Cav1.2 channels during CaM inhibition (Figure 5). Application of W7 increased the number of coupled Cav1.2 channel events in PKCα null (PKCα^−/−; n=5 cells), but not in AKAP150 null myocytes (AKAP150^−/−; n=9 cells; P<0.05). Because the expression of PKCα is similar in WT and AKAP150^−/− myocytes,⁹ the lack of effect of W7 on Cav1.2 channels in AKAP150^−/− was not attributable to decreased PKCα expression. These data suggest that CaM antagonists work downstream of PKCα and that AKAP150 participates in the molecular events that facilitate coupled gating of Cav1.2 channels.

Coupling of Cav1.2 Channels Involves Interactions Between Neighboring Channels via Their C Termini

We investigated the role of the C terminus of Cav1.2 channels in coupled gating. To do this, a Cav1.2 construct lacking amino acids 1670 to 2171 (Cav1.2Δ1670X), which eliminates a large section of the C-terminal tail, was expressed in tsA-201 cells (Figure 6A). These channels lack the putative AKAP-binding region.²⁴ Unlike WT channels, Cav1.2Δ1670X channels showed no coupled gating activity under either control conditions or after the application of W7 (n=5 cells), indicating that residues 1670 to 2171 of Cav1.2 are critical for coupled gating activity.

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Figure 6. Coupled gating involves the C tail of Cav1.2 channels. A, Bar plot of number of sites showing coupling of Cav1.2 channels in cells expressing a truncated Cav1.2 channel (Cav1.2Δ1670X) or WT Cav1.2 channels before and after application of 100 μmol/L W7. B, Bar plot of effective FRET between Cav1.2-TS-EGFP and Cav1.2-TS-tRFP or WT Cav1.2-EGFP and Cav1.2-tRFP before and after PDBu or W7. The inset shows an illustration of Cav1.2-EGFP and Cav1.2-tRFP. C, Proposed model for coupled gating of Cav1.2 channels.

The C termini of Cav1.2 channels could be involved in coupled gating by coming into close proximity. To examine this possibility, we expressed WT and TS Cav1.2 channels with EGFP or tRFP fused to their C termini and determined FRET between channels (Figure 6B). Under control conditions, FRET between WT Cav1.2-EGFP and Cav1.2-tRFP was low (0.02±0.02; n=10 cells). This is consistent with the observation of a relatively low frequency of coupled Cav1.2 events under these experimental conditions (see Figure 1 above). Application of W7 or PDBu, which increases coupled gating of Cav1.2 channels, increased FRET between WT Cav1.2-EGFP and Cav1.2-tRFP channels to 0.16±0.02 (n=8 cells) and 0.23±0.03 (n=10 cells), respectively (P<0.05). Interestingly, under control conditions, FRET was higher in cells expressing tRFP- and EGFP-tagged Cav1.2-TS channels (0.10±0.01; n=10 cells) than between WT Cav1.2 channels (P<0.05; Figure 6B). These data suggest that the C termini of Cav1.2 channels come into close proximity under conditions that favor coupled gating.