Ca2+ signaling amplification by oligomerization of L-type Cav1.2 channels Rose E. Dixon, Can Yuan, Edward P. Cheng, Manuel F. Navedo1, and Luis F. Santana2

Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195

Edited by Richard W. Aldrich, University of Texas, Austin, TX, and approved December 15, 2011 (received for review October 11, 2011) 2+ fl Ca in ux via L-type Cav1.2 channels is essential for multiple phys- Cav1.2 channels increases during Timothy syndrome (TS), in- iological processes, including gene expression, excitability, and con- creasing Ca2+ influx, AP duration, and arrhythmogenesis during traction. Amplification of the Ca2+ signals produced by the opening this pathological condition (5, 6). of these channels is a hallmark of many intracellular signaling cas- Although the mechanism underlying concerted openings of cades, including excitation-contraction coupling in heart. Using opto- Cav1.2 channels is unclear, interactions between their C termini (5, genetic approaches, we discovered that Cav1.2 channels form 6, 8, 9) have been hypothesized to enable functional coupling of clusters of varied sizes in ventricular myocytes. Physical interaction these channels. However, the notion that protein-to-protein between these channels via their C-tails renders them capable of co- interactions can render Cav1.2 channels capable of becoming ordinating their gating, thereby amplifying Ca2+ influx and excitation- functionally coupled is controversial (10). To resolve this impor- contraction coupling. Light-induced fusion of WT Cav1.2 channels tant issue, we used a combination of optogenetic, imaging, and with Cav1.2 channels carrying a gain-of-function mutation that electrophysiologial tools to test four hypotheses that should hold causes arrhythmias and autism in humans with Timothy syndrome true if functional coupling of Cav1.2 channels were to occur and 2+ 2+ (Cav1.2-TS) increased Ca currents, diastolic and systolic Ca lev- modulate cardiac function. Accordingly, (i)Cav1.2 channels 2+ els, contractility and the frequency of arrhythmogenic Ca fluctu- should be found sufficiently close to each other on the surface ations in ventricular myocytes. Our data indicate that these membrane to permit physical interaction and coupling; (ii) binding changes in Ca2+ signaling resulted from Ca 1.2-TS increasing the v of Cav1.2 channels via their C termini should increase the activity activity of adjoining WT Cav1.2 channels. Collectively, these data and coupling coefficient (κ) of adjoining channels; (iii) physical support the concept that oligomerization of Ca 1.2 channels via v interactions between gain-of-function mutant Cav1.2 channels their C termini can result in the amplification of Ca2+ influx into (e.g., Cav1.2 carrying the single point mutation that causes TS) and excitable cells. WT Cav1.2 channels should increase the activity of the WT channels; and (iv) physical interaction of Cav1.2 channels should EC coupling | voltage-gated calcium channels | coupled gating | 2+ translate into a larger increase in [Ca ]i during EC coupling in calcium sparklets ventricular myocytes. Our data suggest that Cav1.2 channels form clusters of varied PHYSIOLOGY n ventricular myocytes, membrane depolarization during the sizes in ventricular myocytes. Physical interaction between Cav1.2 2+ Iaction potential (AP) opens sarcolemmal Cav1.2 channels. Ca channels via their C-tails increased the amplitude of whole- 2+ 2+ influx via a single or small cluster of Cav1.2 channel(s) induces Ca currents. We found that this increase in Ca influx is pro- 2+ 2+ 2+ a local increase in intracellular Ca ([Ca ]i) called a “Ca duced by an increase in the activity of linked channels, which in- ” 2+ sparklet (1). A Ca sparklet activates a small cluster of closely dicates that Cav1.2 channels could modulate the gating of linked apposed ryanodine receptors (RyRs) located in the sarcoplasmic channels via protein-to-protein interactions. Importantly, fusion 2+ reticulum (SR) of these cells via the mechanism of Ca -induced of Cav1.2 channels increased EC coupling in ventricular myo- Ca2+ release (CICR), producing a “Ca2+ spark” (1, 2). The syn- cytes. Based on these findings, we formulated a general model in 2+ 2+ chronous activation of multiple Ca sparks throughout the which oligomerization of Cav1.2 channels amplifies Ca influx myocyte during the AP results in a massive release of Ca2+ from in excitable cells. 2+ the SR that amplifies Ca influx via Cav1.2 channels, causing 2+ aglobalincreasein[Ca ]i that triggers contraction. This chain of Results events is known as excitation-contraction (EC) coupling. Coupled Gating and Clustering of Cardiac Cav1.2 Channels. To begin, At the membrane potentials reached during the plateau of the we show elementary Cav1.2 channel currents (iCa) recorded from ventricular AP (∼+50 mV), the driving force for Ca2+ entry at ventricular myocytes isolated from a WT mouse and a mouse physiological Ca2+ levels (∼2 mM) is so low that the opening of model of TS (5) during a step depolarization to −30 mV (Fig. 1A). fi 2+ asingleCav1.2 channel is not suf cient to raise local [Ca ]i be- The amplitude of iCa was similar in WT (0.49 ± 0.17 pA) and TS yond the threshold of activation of a Ca2+ spark (3). However, as myocytes (0.47 ± 0.05 pA; P = 0.36). We analyzed these currents suggested by Inoue and Bridge (3), the probability of Ca2+ spark using a coupled Markov chain model to determine the κ of the activation during this phase of the AP is very high (i.e., >0.9). To channels underlying them. The κ value could range from 0 for – achieve this, 10 15 Cav1.2 channels must simultaneously open to purely independently gating channels to 1 for channels that always reliably activate a Ca2+ spark (3, 4). However, because at ∼+50 mV the open probability (Po)ofCav1.2 channels is ∼0.5, the probability that 10–15 independently gating channels will open Author contributions: R.E.D. and L.F.S. designed research; R.E.D., C.Y., E.P.C., and M.F.N. simultaneously is 0.510–0.515, which raises a fundamental question: performed research; C.Y. contributed new reagents/analytic tools; R.E.D., E.P.C., and M.F.N. analyzed data; and R.E.D., E.P.C., and L.F.S. wrote the paper. If the probability of coincident openings of 10–15 Cav1.2 channels is so low, why is the probability of Ca2+ spark activation during the The authors declare no conflict of interest. AP >0.9? A potential answer to this conundrum is suggested by This article is a PNAS Direct Submission. work from our laboratory (5, 6) and others (3, 7), suggesting that Freely available online through the PNAS open access option. 2+ small clusters of voltage-gated Ca channels can be functionally 1Present address: Department of Pharmacology, University of California, Davis, CA 95616. coupled, leading to concerted openings of adjacent channels, and 2To whom correspondence should be addressed. E-mail: [email protected]. fi 2+ fl thus the necessary ampli cation of Ca in ux during EC cou- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pling. Interestingly, the probability of coupled gating between 1073/pnas.1116731109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1116731109 PNAS | January 31, 2012 | vol. 109 | no. 5 | 1749–1754 Downloaded by guest on September 26, 2021 A B FKF1 undergoes a conformational change upon illumination with (i) κ=0.00(ii) κ=0.18 0.08 blue light (473 or 488 nm), which permits binding to GI. We fused *** C C κ) Cav1.2 channels to FKF1 or GI on their C-tails. These proteins O O 1 1 0.06 were linked to the C-tails of the channels for three reasons. First, κ O2 =0.19 the C terminus of Cav1.2 channels has been shown to dimerize C κ=0.12 in vitro (8). Second, FRET experiments have suggested that O C 0.04 1 D O O Cav1.2 channels can physically interact via their C-tails (Fig. 1 ) 2 1 O O (6). Third, Ca 1.2 channels lacking most of their C termini do not 3 2 0.02 v O fi 3 display coupled gating behavior (6). Because the ef ciency of

O Coupling coefficient ( 4 light-induced fusion depends on the relative abundance of Ca 1.2- O 0.00 v 5 0.1 s 0.5 pA WT TS GI and Cav1.2-FKF1 channels, we expressed a mutant Cav1.2-GI C tsA-201 cells Cardiomyocytes D channel that is insensitive to dihydropyridines (DHP) (14) to 50 CaV1.2-WT CaV1.2-TS CaV1.2-TS 0.08 quantify the contribution of each channel to the macroscopic *** current. We performed experiments in tsA-201 cells in which 40 0.06 ∼50% of their macroscopic Cav1.2 current (ICa)wasDHPin- sensitive (56 ± 14% I :44± 14% I ). 30 CaV1.2-GI CaV1.2-FKF1 0.04 A testable prediction of the hypothesis that 473-nm light induces 20 fusion of Cav1.2-FKF1 and Cav1.2-GI channels is that the diffusion 0.02 coefficient (D) of randomly moving channels should decrease 10 % of mobile pixels upon fusion of multiple channels (15). Thus, we used raster image Mean FRET efficiency Mean FRET 0 0.00 correlation spectroscopy (RICS) (16) to determine D of tRFP- 12345 12345 12345 -TS tagged Ca 1.2-GI and Ca 1.2-FKF1 channels before and after Size of aggregate -WT v v WT TS light-induced fusion. Consistent with our hypothesis, we found that D ± Fig. 1. Ca 1.2 channels undergo coupled gating and cluster in cell mem- of Cav1.2-GI and Cav1.2-FKF1 channels decreased from 2.2 v μ 2 ± μ 2 n P < branes. (A)iCa recordings and their κ values obtained from ventricular 0.1 m /s under control conditions to 1.2 0.1 m /s ( =6; myocytes isolated from WT (i) or transgenic mice with the TS mutation (ii) 0.05; Fig. 2 A–E) after exposure to 473-nm light, suggesting suc- during 2-s step depolarizations from −80 to −30 mV. Openings are shown as cessful light-induced fusion of these channels. Fig. 2F shows the fl downward de ections, with the closed state denoted by C, and the open effects of Cav1.2-GI and Cav1.2-FKF1 fusion on ICa in a repre- state of 1–5 channels by O. (B) Bar plot of mean κ values from WT and TS sentative tsA-201 cell. Fusion of these channels resulted in ventricular myocytes. (C) Bar chart showing the relative percent of mobile a ∼twofold increase in the amplitude of ICa at +20 mV. Appli- pixels in tsA-201 cell membranes (Left) or ventricular myocyte Z-lines (Right) cation of the DHP nifedipine (1 μM), which immobilizes the that were occupied by the stated size of aggregates of tRFP-tagged Cav1.2- voltage sensor and blocks Cav1.2 channels (17), decreased the WT or Cav1.2-TS channels (n = 5 for each). (D) Bar plot of mean FRET effi- amplitude of ICa by ∼50%, suggesting that Cav1.2-GI and Cav1.2- ciency between Cav1.2-WT-tRFP and Cav1.2-WT-EGFP (n = 12) or Cav1.2-TS- tRFP and Cav1.2-TS-EGFP (n = 7). ***P < 0.001. FKF1 channels were expressed to a similar extent in this cell. The current–voltage (I–V) relationship for five such cells is summa- rized in Fig. 2G. Further analysis of ICa revealed that fusion of fi gate together. In Fig. 1A, i, we present iCa records produced by the Cav1.2-GI and Cav1.2-FKF1 also caused a signi cant leftward shift ± ± P stochastic activation of one Cav1.2 channel (κ = 0.00; Upper)or (control V1/2 =13.5 1.0 mV; light V1/2 = 7.0 0.9 mV; = the concerted activation of multiple channels (κ = 0.19; Lower)in 0.028) in the voltage dependence of the normalized conductance H a WT myocyte. Fig. 1A, ii, shows two records of iCa produced by (G/Gmax)ofICa (Fig. 2 ), suggesting an increase in the voltage concerted opening of 2–5 channels in ventricular myocytes from sensitivity of these channels. Interestingly, fusion of Cav1.2-GI and fi transgenic mice expressing Cav1.2-WT and Cav1.2-TS channels. Cav1.2-FKF1 channels signi cantly increased the steepness factor fi ± The κ and frequency of coupled Cav1.2 channel events was higher of the Boltzmann function used to t the G/Gmax data from 7.3 − in TS than in WT myocytes (Fig. 1B; P = 2.33 × 10 6). 0.8 mV to 6.4 ± 0.7 mV (P = 0.002; Table S1), suggesting an We used the number and brightness (N&B) analysis (11) to test increase in the cooperativity of these channels. None of these the hypothesis that Cav1.2 channels should come close together changes in ICa were observed in tsA-201 cells expressing either within a particular volume in the surface membrane so that CaV1.2-GI or Cav1.2-FKF1 alone (Figs. S2 and S3 and Table S1). physical interaction and functional coupling is likely (Fig. 1C). 2+ fl N&B is a moment analysis that determines the aggregation state of Fusion of Cav1.2 Channels Increases Ca In ux by Increasing the κ fluorescent proteins—in our case, Ca 1.2 channels fused to the tag Activity and Between Adjacent Channels. ICa is related to the v N red fluorescent protein (tRFP) (12)—based on the assumption number ( ) of functional Cav1.2 channels in the surface mem- P that fluorescence intensity fluctuations within individual pixels are brane, their o, and the amplitude of their unitary currents (iCa) N·P · caused by occupation number. Using this analysis, clusters of up to by the equation ICa = o iCa. Thus, we investigated whether N P five Cav1.2-WT and Cav1.2-TS channels were detected in tsA-201 a change in , o,oriCa contributed to the increase in ICa ob- cells and ventricular myocytes. Consistent with this finding, in tsA- served after fusion of Cav1.2-GI and Cav1.2-FKF1 channels. First, 201 cells, we detected FRET between WT or TS Cav1.2 channels we examined whether the number of Cav1.2-GI and Cav1.2-FKF1 fused to the EGFP or tRFP at their C termini (Fig. 1D), suggesting changed after light-induced fusion of these channels in tsA-201 that Cav1.2 channels can and do come within close proximity of cells using two complementary approaches: RICS and gating one another. currents. RICS has been used to determine the concentration of fluorescent proteins (16). Likewise, gating currents can be used to Light-Induced Fusion of Cav1.2 Channels Alters the Voltage Dependence determine the number of Cav1.2 channels using the following 2+ of Activation and Amplitude of Whole-Cell Ca Currents. We next in- rationale. The time integral of the ON gating charge (Qon)is vestigated the hypothesis that Cav1.2 channels gate coordinately proportional to the number of voltage-gated channels in the via physical interaction between C-tails using a light-activated surface membrane (Qon = N·q,whereq is the number of ele- fusion system (13) that links closely apposed C-tails of Cav1.2 mentary charges per channel) and is independent of iCa and Po. channels as illustrated in Fig. S1. The light-activated fusion system Thus, assuming q does not change, Qon can be used to determine involves FLAVIN-BINDING, KELCH REPEAT, F BOX 1 whether a change in ICa is associated with changes in the number (FKF1) and GIGANTEA (GI) proteins of Arabidopsis thaliana. of Cav1.2 channels (18).

1750 | www.pnas.org/cgi/doi/10.1073/pnas.1116731109 Dixon et al. Downloaded by guest on September 26, 2021 A (i) B (i) C D =2.29 μm2/s D D = 1.45 μm2/s E 2.5

5 5 2.0 0 0 5 5 -10 -10 /s) 1.5 (ii) (ii) 2 *** -3 -3 1.0 Fig. 2. Fusion of Cav1.2 channels alters ICa.(A and B) RICS 8 8 D (μm analysis in an exemplar tsA-201 cell expressing Cav1.2-GI-tRFP 4 4 0.5 (iii) (iii) and Ca 1.2-FKF1-tRFP before (A) and after (B) fusion. The 0 42 0 42 v G(x,y) x 10 35 G(x,y) x 10 35 0.0 regions of interest outlined in i are magnified in ii, and the 21 28 28 Pixels 21 28 28 Pixels 35 21 35 21 autocorrelations calculated from these regions are shown in Pixels 42 Pixels 42 Fused Control iii.(C and D) Fit of the correlation functions with resultant F GH +20 mV diffusion coefficient (D) values indicated above. (E) Bar plot -60 -40 -20 0 20 40 60 80 1.0 Control 0.0 -80 Fused of D values obtained from six such tsA-201 cells before and after fusion. (F) Representative ICa records from a tsA-201 cell

max transfected with Cav1.2-FKF1 and DHP-insensitive Cav1.2-GI 0.5 during a 300-ms depolarizing step to +20 mV from the holding G/G Control potential of −80 mV before (black) and after fusion (blue). (G) (WT-FKF1 + DHPins-GI) (normalized) I–V plot summarizing the results from n = 5 such cells with Ca I 20 pA/pF Control 0.0 voltage steps from −80 mV to test potentials ranging from −60 Fused -1.0 Fused -60 -40 -20 0 20 40 (WT-FKF1 + DHPins-GI) 50 ms mV to +70 mV. Currents were normalized to the peak. (H) Voltage I J K dependence of G/G before and after fusion fitted with (i) ICa 1.5 0.6 max ** Boltzmann functions. Conductance (G) was normalized to the maximum conductance (Gmax). (I)ICa (i) recorded in a tsA-201 1.2] (μM)

0.4V cell expressing Cav1.2-FKF1 and Cav1.2-GI before (black) and 1.0 after fusion (blue) during a 20-ms step to +20 mV from the 50 pA/pF − 0.2 holding potential of 90 mV. Gating current (Ig)(ii) recorded (ii) I at E g Ca 5 ms in the same cell during 20-ms steps to ECa.(J) Bar plot sum- marizing the results from n = 5 such cells. (K) Bar plot showing Fold change after fusion 0.5 0.0 20 pA/pF [tRFP-tagged Ca mean concentrations of tRFP-tagged Cav1.2-FKF1 and Cav1.2- Q on GI channels in the confocal volume, before and after fusion. 2 ms , Peak ControlFused I Ca Error bars indicate SEM. ***P < 0.001; **0.001 < P < 0.01.

2+ We recorded Qon during step depolarizations to the reversal (34 ± 1vs.37± 1 nM, P > 0.05), it increased Ca sparklet activity potential (ECa)ofICa. The ICa evoked by voltage steps from ∼2.9-fold and the κ of the Cav1.2 channels underlying these events a holding potential of −90 mV to potentials ranging from −60 to by ∼18.1-fold (Fig. 3 D–F). The increase in Ca2+ influx following +110 mV was used as an index of Cav1.2 channel activity. Al- light-induced fusion of Cav1.2 channels resulted from an increase though fusion of Cav1.2-GI and Cav1.2-FKF1 channels increased in the activity and κ of previously active sites and the emergence of

2+ PHYSIOLOGY peak ICa ∼1.3-fold (n =5,P = 0.007), Qon was similar before and new, coupled, high-activity Ca sparklet sites. P I J 2+ after fusion of these channels ( = 0.46; Fig. 2 and ). RICS In combination, our RICS, QON,ICa,iCa,andCa sparklets analysis of tRFP-tagged Cav1.2-GI and Cav1.2-FKF1 channels data suggest that fusion of Cav1.2 channels does not change the indicated that the concentration of these channels in the surface expression (N) of these channels or the amplitude of their unitary n membrane was similar before and after light-induced fusion ( = currents. Rather, light-induced fusion of Cav1.2 channels increases 2+ 6, P = 0.18; Fig. 2K). These data suggest that the increase in ICa Ca influx by increasing the coupling strength and activity of observed after light-induced fusion is not associated with an in- these channels. crease in the number of Cav1.2 channels. 2+ fi 2+ fl Next, we recorded iCa from cell-attached patches as well as Ca Ampli cation of Ca In ux by Fusion of Cav1.2-TS and Cav1.2-WT sparklets before and after the induction of channel fusion with 488- Channels. An important implication of the data described above P nm light (Fig. 3). Cav1.2 channel openings were evoked by a voltage is that physical interactions between high o Cav1.2 channels and P 2+ fl step from −80 to −30 mV. Fig. 3A shows iCa records from a rep- low o channels could amplify Ca in ux by increasing the open resentative tsA-201 cell patch expressing Cav1.2-GI and Cav1.2- probability of the low-activity channels. For example, fusion of FKF1 before and after illumination with 488-nm light. Although genetically or posttranslationally modified channels produced by light-induced fusion of these channels did not alter the amplitude either a genetic mutation such as Cav1.2-TS or by phosphoryla- of elementary Ca2+ currents (0.51 ± 0.01 pA vs. 0.51 ± 0.01 pA; tion during signaling with unmodified WT channels could amplify 2+ fl P = 0.70), it increased the activity (i.e., NPo)oftheCav1.2 channels the effects of a mutation or signaling pathway on Ca in ux. in this patch 17.8-fold. On average, NPo increased from 0.01 ± Thus, we hypothesized that high-activity Cav1.2-TS channels −16 0.001 to 0.20 ± 0.02 (P = 1.22 × 10 ) following light-induced could increase the activity of an adjoining Cav1.2-WT channel. To fusion of Cav1.2-GI and Cav1.2-FKF1, with some individual test this hypothesis, the effects of light-induced fusion of Cav1.2- patches having NPo values ≤0.9 following fusion (Fig. 3B). Before TS-FKF1 and WT Cav1.2-GI (DHP-insensitive) channels on ICa the induction of light-induced fusion, the mean κ was 0.01 ± 0.003, were investigated in tsA-201 cells (Fig. 4 A–D). Under control suggesting that openings were produced by the stochastic activa- conditions, Cav1.2-GI and Cav1.2-TS-FKF1 channels produced tion of single Cav1.2 channels (Fig. 3C). In the patch in Fig. 3A, robust ICa records with a slowly inactivating, sustained component light-induced fusion of Cav1.2-GI and Cav1.2-FKF1 increased the characteristic of currents produced by Cav1.2-TS (19, 20) (Fig. likelihood of observing larger amplitude currents resulting from 4A). Fusion resulted in an increase in the amplitude of peak and the simultaneous opening of 2–5 channels, increasing the κ of the sustained components (measured 250 ms after the onset of de- channels in this patch from 0.00 to 0.16 (Fig. 3A, ii). On average, polarization) of ICa (Fig. 4B). Interestingly, the increase in the fl light-induced fusion of Cav1.2-GI and Cav1.2-FKF1 increased the sustained component was not simply a re ection of the increase in coupling strength between these channels ∼10-fold (i.e., κ in- the peak current but rather the increase was nonscalar as can be −18 creased from 0.01 ± 0.003 to 0.10 ± 0.01; Fig. 3C; P = 9.13 × 10 ). observed when we normalize the ICa to their peaks as in Fig. 4B, Consistent with these data, although fusion of Cav1.2 channels Inset. After we applied 1 μM nifedipine to immobilize the voltage 2+ did not alter the amplitude of elementary Ca sparklet events sensor of Cav1.2-TS-FKF1 channels, we decreased the amplitude

Dixon et al. PNAS | January 31, 2012 | vol. 109 | no. 5 | 1751 Downloaded by guest on September 26, 2021 (i) Control (ii) Fused were fused. Supporting this hypothesis, we detected a significant A nP =0.04 nP =0.71 BC κO κO 1.0 0.3 ± ± P =0.00 =0.16 *** *** shift in V1/2 from 10.4 1.8 mV to 1.6 1.7 mV ( = 0.012; Fig. C 0.8 D O 4 and Table S1) and an increase in the steepness factor of the G/ 1 0.2 O2 – ±

O 0.6 Gmax voltage relationship from 10.2 1.6 mV in control con- O3 O

NP ± 4 0.4 ditions to 8.1 1.5 mV after fusion of Cav1.2-GI and Cav1.2-TS- O5 O 0.1 P n 6 FKF1 channels ( = 0.044; = 5 cells). Note that the V1/2 ob- 50 ms 0.5 pA 0.2 served upon fusion of these channels shifted toward the V1/2 seen 0.0 ( κ ) Coupling coefficient 0.0 in cells expressing Cav1.2-TS-FKF1 alone (∼−6 mV; Table S1). ControlFused ControlFused Importantly, blue light did not produce any of these changes to (i) Control (ii) Fused D EF ICa in tsA-201 cells expressing Cav1.2-TS-FKF1 alone (Figs. S2C κ=0.00 κ=0.31 2.5 *** 0.8 O E F 9 1 s *** and S3 and and Table S1). O 2.0 8 0.6 50 nM A similar experiment was performed, though in tsA-201 cells O7 O S 1.5 6 0.4 expressing WT Cav1.2-GI and DHP-insensitive Cav1.2-TS-FKF1 O NP 5 1.0 channels (Fig. 4E). This gave us the opportunity to investigate O 4 0.2 O3 0.5 whether inhibition of WT channels would alter the activity of

O2 0.0 ( κ ) Coupling coefficient 0.0 adjoined TS channels. As expected, the peak and sustained com- O1 C rol ponent of ICa increased (1.4-fold-and 2.9-fold, respectively) fol- ControlFused Cont Fused lowing light-induced fusion of Cav1.2-GI and Cav1.2-TS-FKF1

Fig. 3. Single-channel analysis of fused Cav1.2. (A) Representative iCa channels. The nonscalar increase in the sustained component recordings obtained from a tsA-201 cell transfected with Cav1.2-GI and Cav1.2- compared with the peak can be observed when we normalize the − − κ FKF1 during a 2-s step depolarization from 80 to 30 mV (i) before ( =0)and ICa to their peaks as in Fig. 4F, Inset. Application of nifedipine to (ii)after(κ = 0.16) light-activated fusion. (B and C) Box-and-whisker plots of the selectively inhibit Ca 1.2-GI channels decreased the sustained κ v mean NPo (B) and (C) of multiple records before and after fusion (n = 5 cells). component of I back to control levels (Fig. 4 E and F). Whiskers show the maximum and minimum values; + indicates the mean value; Ca boxes indicate the first, second, and third quartile values. (D) Optical record- Next, we investigated whether this increase in ICa was due to 2+ ings of Ca influx via Cav1.2 channels (i) before (κ = 0) and (ii) after fusion (κ = changes in the number of Cav1.2 channels in tsA-201 cells using 0.31). (E and F) Box-and-whisker plots of the mean NPs (E) and κ (F) of multiple Qon and RICS as described above. Fusion of Cav1.2-GI and < sparklet recordings before and after fusion. ***P 0.001. Cav1.2-TS-FKF1 channels increased peak ICa by ∼1.3-fold (P = 0.002) without a concomitant change in Qon (n =5,P = 0.14; Fig. 4 G and H). Consistent with this finding, RICS analysis of tRFP- of I and eliminated the slow, inactivating component of the Ca tagged Cav1.2-GI and Cav1.2-TS-FKF1 channels indicated that the current (Fig. 4 A and B). These data are consistent with the hy- concentration of these channels within the confocal volume ex- pothesis that the increase in the sustained component of ICa amined was similar before and after light-induced fusion (n =5; following light-induced fusion of Cav1.2-GI and Cav1.2-TS-FKF1 P = 0.09; Fig. 4I). These findings suggest that the increase in the was due to a fraction of the WT channels opening late during the noninactivating component of ICa was due to an increase in the depolarization pulse similar to the TS channels to which they activity of WT Cav1.2-GI channels late in the depolarization pulse,

Fig. 4. Fusion of Ca 1.2-WT with Ca 1.2-TS confers TS A +20 B I , Peak I , 250 ms C mV v v 0 Ca Ca -60 -40 -20 0 20 40 60 80 properties onto WT channels. (A) Representative whole- 0 -80 Nifedipine cell currents obtained from a tsA-201 cell transfected with (DHPins-GI) -10 DHPins Control Cav1.2 -GI and Cav1.2-TS-FKF1 during a voltage step − -20 Fused from 80 to +20 mV before (black) and after fusion (blue) Nifedipine and after subsequent application of 1 μM nifedipine (red). -30 (normalized) (B) Bar plot showing the peak I at +20 mV obtained from Control Ca Ca

(DHPins-GI + TS-FKF1) (pA/pF at +20mV) -40 the cell shown in A and the remaining noninactivating ICa Ca I Control − Peak I after 250 ms of a 300-ms depolarizing pulse from 80 to Fused 50 ms 10 pA/pF -50 -1 Fused (DHPins-GI + TS-FKF1) +20 mV. (BInset) Currents from A, normalized to their I , Peak I , 250 ms peaks to show the nonscalar increase in the non- D 1.0 E +20 F Ca Ca Control 0 inactivating component. (C)I–Vplotsummarizingthe Fused -80 − Nifedipine Control results from n = 5 such cells with voltage steps from 80 mV (DHPins-TS-FKF1) Fused to test potentials ranging from −60 to +70 mV. Currents max -10 0.5 Nifedipine were normalized to the peak. (D) Voltage dependence of G/G G/Gmax before and after fusion, fitted with Boltzmann Control -20 (DHPins-TS-FKF1 + WT-GI) functions. (E) Whole-cell currents from a tsA-201 cell

(pA/pF at +20mV) DHPins

Ca transfected with Cav1.2 -TS-FKF1 and Cav1.2-GI. (F)Bar I

0.0 10 pA/pF plot summarizing data from the cell shown in E.PeakICa -60 -40 -20 0 20 40 Fused 50 ms -30 mV (DHPins-TS-FKF1 + WT-GI) and ICa remaining after 250 ms of a 300-ms pulse to +20 mV (i) I are plotted for control, fused, and nifedipine conditions. (F G Ca H 1.5 I 0.4 Inset)CurrentsfromE, normalized to their peaks to show ** the nonscalar increase in the noninactivating component. 0.3

1.2] (μM) (G)ICa (i) recorded in a tsA-201 cell expressing Cav1.2-FKF1 V and Cav1.2-GI before (black) and after (blue) fusion during 50 pA/pF 1.0 0.2 a 20-ms step to +20 mV from the holding potential of −90

5 ms mV. Gating current (Ig)(ii) recorded in the same cell during (ii) Ig at ECa 0.1 20-ms steps to ECa.(H) Bar plot summarizing the results Fold change after fusion from n = 5 such cells. (I) Bar plot showing mean concen- 0.5 [tRFP-tagged Ca 0.0 10 pA/pF on Q trations of tRFP-tagged Cav1.2-TS-FKF1 and Cav1.2-GI , Peak ControlFused 2 ms I Ca channels in the confocal volume, before and after fusion. Error bars indicate SEM. **0.001 < P < 0.01.

1752 | www.pnas.org/cgi/doi/10.1073/pnas.1116731109 Dixon et al. Downloaded by guest on September 26, 2021 2+ (i) Untransfected Finally, we recorded [Ca ]i and cell length in myocytes A Control Fused expressing Cav1.2-TS-FKF1 and WT Cav1.2-GI channels. The 2+ M amplitudes of the AP-evoked [Ca ] transients and contractions n i 0

0 under control conditions (i.e., before illumination with 473 nm) 1 s 1 (ii) were larger than in untransfected myocytes and cells expressing m μ WT Ca 1.2-FKF1/Ca 1.2-GI channels (Fig. 5C, i). Fusion of WT 4 v v (i) Ca 1.2-FKF1 & Ca 1.2-GI P B V V and mutant TS channels induced a further increase in systolic ( = 2+ 0.004) and diastolic [Ca ]i levels (P = 0.007) and produced

M ∼ P n D– n a 1.7-fold increase in cell shortening ( = 0.003; =6;Fig.5 0

0 F (ii) 1 s 1 ). Furthermore, fusion of these channels increased the frequency of arrhythmogenic spontaneous SR Ca2+ release events (Fig. 5C, m μ ii 2+ C iii 4 ) and [Ca ]i alternans (Fig. 5 , ). Collectively, these data 2+ suggest that fusion of Cav1.2 channels could increase [Ca ]i and

(i) CaV1.2-TS-FKF1 & CaV1.2-GI (ii) cell shortening during EC coupling and induce arrhythmogenic C 2+ changes in [Ca ]i. M

n Discussion 0 0

1 s 1 Our N&B, FRET, optogenetic, and electrophysiological data (iii) support a unique model for the spatial organization and func- tional regulation of Cav1.2 channels in ventricular myocytes.

M –

n N&B analysis suggests that Cav1.2 channels form clusters of 2 5 0

0 channels that diffuse together within the surface membrane of 1 s 1 i i ] ** ] ** ** heterologous cells as well as ventricular myocytes. Not only can

2+ 2.0 1.4 2.5

DFE 2+ 1.2 ** ** these channels cluster, but, as our FRET data indicate, they * 2.0 1.5 1.0 come close enough to physically interact via their C-tails. The 0.8 1.5 observation that we can fuse the C termini of these channels 1.0 0.6 1.0 supports the view that Cav1.2 channels cluster and can form 0.5 0.4 relatively stable protein-to-protein interactions, which is consis- 0.5 0.2 tent with the observation of crystals of Cav1.2 C termini (8) and Fold change: systolic [Ca Fold change: cell shortening Fold change: diastolic [Ca 0.0 0.0 0.0 Cav1.2 dimers using electron microscopy (9). These interactions allow Cav1.2 channel oligomers to open in concert. On the basis Untrans.WT/WTWT/TS Untrans.WT/WTWT/TS Untrans.WT/WTWT/TS of these data, we propose a model in which oligomerization of 2+ Cav1.2 channels via their C-tails constitutes a unique general Fig. 5. Ca signaling in ventricular myocytes is altered by Cav1.2 fusion. fi 2+ fl Evoked Ca2+ transients (i) and simultaneous cell length changes (ii) from (A) mechanism for the ampli cation of Ca in ux in excitable cells. We propose that when Cav1.2 channels bind to a neighboring untransfected and (B) ventricular myocytes transfected with Cav1.2-GI and PHYSIOLOGY 2+ Cav1.2-FKF1. (C)Ca transients from myocytes expressing Cav1.2-TS-FKF1 channel via their C-tails, they become allosteric activators of ad- and Cav1.2-GI before (i) and after (ii and iii) fusion. (C, ii Inset) Entire re- joining channels. Binding of a Cav1.2 channel to a neighboring cording from which a section (highlighted in gray) is shown below with an channel increases Ca2+ influx through two complementary expanded time scale. Cells were paced at a frequency of 1 Hz before (Left) mechanisms. First, the C-terminal-to-C-terminal binding increases and after (Right) light-activated fusion. Arrowheads indicate the timing of 2+ the positive cooperativity between Cav1.2 channels. Second, the the stimulations. For comparison, the dashed line shows the diastolic [Ca ]i – increase in cooperativity between Cav1.2 channels promotes before fusion. (D F) Bar plots summarizing results from multiple ventricular a switch from a predominantly stochastic gating mode (i.e., κ ∼0.0) myocytes (untransfected, n = 5; WT/WT, n = 5; WT/TS, n = 6), showing the mean fold-change after fusion (relative to levels before fusion) in diastolic to a mode in which the probability of coupled openings between 2+ 2+ κ ∼ – [Ca ]i, systolic [Ca ]i, and cell shortening, respectively. **0.001 < P < 0.01; interacting channels is relatively high (i.e., 0.1 0.3). Accord- *0.01 < P < 0.05. ingly, multichannel openings of adjoined Cav1.2 channels results from the coordinated opening of tethered channels and random, coincident activation of independently gating channels. and that inhibition of these channels does not close adjoining If Cav1.2 channels bound together via their C-tails exhibit Cav1.2 channels. positive cooperativity, what accounts for the observation that Cav1.2 channels fused via their C-tails do not exhibit fully con- Fusion of Cav1.2 Increases EC Coupling in Ventricular Myocytes. We certed openings and closings? We propose the following hy- investigated the physiological implications of coupling of Cav1.2 pothesis to explain our observation. The physical coupling channels on EC coupling. To do this, -evoked between Cav1.2 channels’ C-tails results in a low level of positive 2+ [Ca ]i transients and cell shortening were recorded in adult rat cooperativity between Cav1.2 channels. Accordingly, tethered ventricular myocytes expressing Cav1.2-GI and Cav1.2-FKF1 chan- Cav1.2 channels become loosely coupled such that the confor- nels before and after light-induced fusion of these channels (8). mational change associated with the opening of one Cav1.2 Myocytes were loaded with the fluorescent Ca2+ indicator Rhod-2 channel does not necessarily translate into the opening of 2+ 2+ to image [Ca ]i. As a control experiment, we recorded [Ca ]i and adjoined channels. Results of our analyses of the increase in NPo cell length in untransfected ventricular myocytes before and after and the increase in κ after fusion support our hypothesis. Though illumination with 473-nm light. In these cells, exposure to 473-nm the fusion of Cav1.2 channels consistently translated into an in- light did not alter the diastolic (151 ± 3 nM vs. 155 ± 11 nM, P = crease in κ and activity, it never increased κ to ∼1. 2+ ± ± P 0.68), the systolic [Ca ]i (329 51 nM vs. 312 47 nM, = 0.14), The increase in the noninactivating, Cav1.2-TS–driven compo- ± μ or the amplitude of the accompanying contraction (2.8 0.7 mvs. nent of ICa after the fusion of Cav1.2-GI to high-activity Cav1.2-TS- 3.1 ± 0.8 μm, P = 0.34; n =5;Fig.5A and D–F). In ventricular FKF1 channels also supports our hypothesis of positive coopera- myocytes expressing Cav1.2-FKF1 and Cav1.2-GI, light-induced tivity. Because light per se does not alter the activity or iCa of FKF1 2+ fusion of these channels caused an increase in diastolic [Ca ]i or GI-fused channels, what caused this increase in the late ICa when P 2+ P P ( = 0.04), systolic [Ca ]i ( = 0.007), and amplitude ( =0.003) WT Cav1.2 channels are inactivated? Our data suggest an answer of the accompanying contraction (Fig. 5 B and D–F; n =5). to this intriguing question. We found that immobilization of the

Dixon et al. PNAS | January 31, 2012 | vol. 109 | no. 5 | 1753 Downloaded by guest on September 26, 2021 voltage sensor of Cav1.2-WT channels with nifedipine following gating activity between Cav1.2-WT and Cav1.2-TS channels in light-induced fusion of WT Cav1.2-GI to DHP-insensitive Cav1.2- ventricular myocytes. Indeed, AKAP150 is required for the de- TS-FKF1 channels eliminated the increase in the noninactivating fective Cav1.2 inactivation, action potential prolongation, and component of ICa.Thisfinding suggests that Cav1.2-WT channels arrhythmias during TS. functioning like TS channels produced the increase in the non- Future studies should address several critical and undetermined inactivating component of ICa after fusion. Furthermore, consistent aspects of our model. For example, what is the mechanism that with our hypothesis above, these data suggest that TS channels can translates C termini–C termini interactions into the opening of open independently of associated WT channels. Future experi- Cav1.2 channels? Furthermore, is AKAP150 a critical determinant ments should examine the molecular mechanisms underlying of the size and stability of Cav1.2 clusters and oligomers as sug- coupling of interacting Cav1.2 channels in detail. gested by the electrophysiological studies described above? Are The observation that fusion of WT Cav1.2 channels to TS other proteins and/or cellular structures involved in these pro- channels makes WT channels function like TS channels has pro- cesses? Since, like protein kinase A, oligomerization of Cav1.2 found implications. First, the observation suggests that within channels shifts the voltage dependence of activation of Cav1.2 to a channel cluster, the channel(s) with the higher open probability more-hyperpolarized potentials, does activation of β-adrenergic (in our case Ca 1.2-TS channels) are likely to determine the level v receptors increase the probability of Cav1.2 channel oligomeriza- of activity of adjoining channels. In humans with TS, an autosomal- tion and coupled gating in ventricular myocytes? Finally, is olig- dominant disease, the effects of Cav1.2-TS (representing only omerization and coupled gating of Ca 1.2 a general phenomenon ∼ 2+ fl fi v 23% of cardiac Cav1.2) on Ca in ux could be ampli ed via in excitable cells? physical interactions with neighboring Cav1.2-WT channels (20). To conclude, we demonstrated that Cav1.2 channels form Thus, expression of a small number of Cav1.2-TS channels could clusters, and that physical interactions of these channels via their 2+ fl have disproportionally large effects on Ca in ux if these chan- C-tails increases the activity and cooperativity of adjoining chan- nels can form stable physical interactions with neighboring nels, thus amplifying Ca2+ influx and consequently EC coupling in WT channels. ventricular myocytes. These findings could have broad implica- An important observation in this study is that fusing Cav1.2 tions. For example, clustering and coupled gating of Ca 1.2 channels increases [Ca2+] and contractility during EC coupling in v i channels in presynaptic sites may enhance the fidelity of neuro- ventricular myocytes. In these cells, Cav1.2 channels and RyRs in transmitter release. Thus, oligomerization of Cav1.2 channels may nearby junctional form a functional unit 2+ 2+ represent a general mechanism for the amplification of Ca influx called a “couplon” (21). Ca influx via Cav1.2 activates small 2+ 2+ in excitable cells. clusters of RyRs via the mechanism of Ca -induced Ca release, 2+ 2+ producing Ca sparks. Because the probability of Ca spark Materials and Methods P occurrence ( Spark) is proportional to the Cav1.2 current and local fl [Ca2+] (22), it is intriguing to speculate that P is likely to be Detailed methods can be found in SI Materials and Methods. Brie y, elec- i Spark trophysiological experiments were performed using standard protocols with higher in couplons with coupled Cav1.2 channels than in couplons an Axopatch 200B amplifier. Optogenetic and imaging experiments were with independently gating channels. Thus, an increase in coupled performed using a Nikon Swept Field or Olympus FV1000 confocal micro- 2+ fl gating of Cav1.2 channels could result in an increase in Ca in ux scope. Fusible Ca 1.2 channel plasmids were generated by tagging Ca 1.2 2+ v v and thus global [Ca ]i. with GI or FKF1 (gifts from Ricardo Dolmetsch, Stanford University School of We used the plant proteins FKF1 and GI to induce physical Medicine, Stanford, CA). Ca2+ sparklets were recorded using a total internal fl fl interactions between Cav1.2 channels fused to these proteins. re ection uorescence microscope (5, 6). Ventricular myocytes were isolated However, in their absence, what promotes Cav1.2–Cav1.2 inter- using standard enzymatic approaches (5, 6). actions, and hence functional coupling, of these channels in ven- tricular myocytes? Two recent studies provide insights into this ACKNOWLEDGMENTS. We thank Drs. A. Vega, A. Barría, and W. Cerpa for technical assistance, and Drs. E. Dickson, J. Mercado, M. Nieves-Cintrón, question (5, 6). It was found that the anchoring protein AKAP150, M. Nystoriak and F. Rieke for reading this manuscript. Support for this work which binds to Cav1.2 channels via leuzine zipper domains located was provided by National Institutes of Health Grants HL085870, HL085686, and in the C termini of both proteins (23), is required for coupled HL098200 and American Heart Association Grants 0735251N and 0840094N.

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