HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 19, 14283-14290, May 11, 2007

This Article Abstract Full Text (PDF) All Versions of this Article: 282/19/14283    most recent M611529200v2 M611529200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Huang, G. Articles by Zeng, W. Search for Related Content PubMed PubMed Citation Articles by Huang, G. Articles by Zeng, W. Social Bookmarking                      What's this?

Ca²⁺ Signaling in Microdomains

Homer1 MEDIATES THE INTERACTION BETWEEN RyR2 AND Cav1.2 TO REGULATE EXCITATION-CONTRACTION COUPLING^*

Guojin Huang, Joo Young Kim, Marlin Dehoff, Yusuke Mizuno, Kristine E. Kamm, Paul F. Worley^¶1, Shmuel Muallem^¶2, and Weizhong Zeng

From the Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390 and Department of Neuroscience, Program in Biochemistry, Cellular, and Molecular Biology, ^¶Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received for publication, December 18, 2006 , and in revised form, February 22, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Excitation-contraction (E-C) coupling and Ca²⁺-induced Ca²⁺ release in smooth and cardiac muscles is mediated by the L-type Ca²⁺ channel isoform Ca_v1.2 and the ryanodine receptor isoform RyR2. Although physical coupling between Ca_v1.1 and RyR1 in skeletal muscle is well established, it is generally assumed that Ca_v1.2 and RyR2 do not directly communicate either passively or dynamically during E-C coupling. In the present work, we re-examined this assumption by studying E-C coupling in the detrusor muscle of wild type and Homer1^-/- mice and by demonstrating a Homer1-mediated dynamic interaction between Ca_v1.2 and RyR2 using the split green fluorescent protein technique. Deletion of Homer1 in mice (but not of Homer2 or Homer3) resulted in impaired urinary bladder function, which was associated with higher sensitivity of the detrusor muscle to muscarinic stimulation and membrane depolarization. This was not due to an altered expression or function of RyR2 and Ca_v1.2. Most notably, expression of Ca_v1.2 and RyR2 tagged with the complementary C- and N-terminal halves of green fluorescent protein and in the presence and absence of Homer1 isoforms revealed that H1a and H1b/c reciprocally modulates a dynamic interaction between Ca_v1.2 and RyR2 to regulate the intensity of Ca²⁺-induced Ca²⁺ release and its dependence on membrane depolarization. These findings define the molecular basis of a "two-state" model of E-C coupling by Ca_v1.2 and RyR2. In one state, Ca_v1.2 couples to RyR2 by H1b/c, which results in reduced responsiveness to membrane depolarization and in the other state H1a uncouples Ca_v1.2 and RyR2 to enhance responsiveness to membrane depolarization. These findings reveal an unexpected and novel mode of interaction and communication between Ca_v1.2 and RyR2 with important implications for the regulation of smooth and possibly cardiac muscle E-C coupling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
The Homer family of scaffolding proteins consists of three members, Homer1, Homer2, and Homer3, and several splice variants (1). The Homers are typified by an N-terminal Ena/VASP homology 1 (EVH)³ domain and C-terminal coiled coil and leucine zipper domains. The EVH domain is a protein-protein binding module that recognizes the proline-rich motifs PPXXF, PPXF, and LPSSP (2), whereas the coiled coil domain and leucine zippers serve to multimerize the Homers into scaffolds that assemble signaling proteins into complexes (1). The Homer1 gene is unique in that it codes for the short Homer1a (H1a) and a long Homer1b/c (H1b/c) (3). H1a lacks the coiled coil domain and leucine zipper, and it antagonizes the functions of the multimerizing Homers by dissociating the complexes formed by them.

Many Ca²⁺ signaling proteins express Homer binding sequences, including G protein-coupled receptors, plasma membrane Ca²⁺ ATPase pumps, IP₃ receptors, canonical transient receptor potential (TRPC) channels, and notably, the ryanodine receptors (RyRs) and the 1C subunit of Ca_v1.2 and 1D subunit of Ca_v1.3 L-type Ca²⁺ channels (1, 2, 4-7). The Homers recruit the Ca²⁺ signaling proteins to the plasma membrane (8) and cellular microdomains, such as dendritic spines (9), but are not essential for assembly or retention of the complexes, because deletion of the Homers does not affect localization of the complexes (10). On the other hand, the Homers modulate the efficiency (2) and intensity of Ca²⁺ signaling (10) by negatively regulating the activity of proteins within the Ca²⁺ signaling complex.

Another important function of Homer1 is the mediation of the conformational coupling between IP₃Rs and TRPC channels. Thus, Homer1 binds TRPC channels and the IP₃Rs to allow the gating of TRPC channels by IP₃Rs (2, 11). This form of coupling is reminiscent of excitation-contraction (E-C) coupling in muscle, which is mediated by the sarcolemmal voltage-activated L-type Ca²⁺ channels and the RyRs Ca²⁺ release channels in the sarcoplasmic reticulum (12-14). Homer1 may play a role in E-C coupling, because it was reported to activate the skeletal and cardiac muscle RyR isoforms RyR1 (15-17) and RyR2 (18), respectively. Furthermore, recently it was suggested that the short, dissociating H1a activates L-type Ca²⁺ current in neocortex pyramidal cells (19).

There are three forms of E-C coupling, depending on muscle type (12-14). Skeletal muscle displays mechanical coupling between the skeletal muscle L-type Ca²⁺ channel isoform Ca_v1.1 and RyR1, whereby the Ca²⁺ release units are organized in tetrads mediated by physical interaction between the cytosolic domains of RyR1 and Ca_v1.1 (12). The activating depolarization is sensed by Ca_v1.1 and is directly conveyed to RyR1 to initiate Ca²⁺ release that then propagates by a Ca²⁺-induced Ca²⁺ release (CICR) mechanism. In cardiac muscle, RyR2 communicates with the cardiac muscle L-type Ca²⁺ channel Ca_v1.2, but the channels do not physically interact. CICR is initiated by Ca²⁺ influx through Ca_v1.2 that then activates RyR2 (13). However, at the cardiac Ca²⁺ release units, RyR2 and Ca_v1.2 are in close proximity, and Ca²⁺ entering through Ca_v1.2 is rapidly sensed by RyR2 (12, 13). Although all RyR isoforms were reported to be expressed in smooth muscle, RyR2 is the major isoform in this muscle type (20), including the urinary bladder detrusor muscle (21). Ca²⁺ influx during E-C coupling of smooth muscle is mediated by Ca_v1.2 (22). However, E-C coupling in smooth muscle was termed loose coupling (14) based on the relatively slow rate of activation of CICR (23).

The properties of E-C coupling and CICR in cardiac and smooth muscles led to the belief that Ca_v1.2 and RyR2 do not directly communicate either passively or dynamically during E-C coupling (12-14, 24). We re-examined this assumption by studying E-C coupling in the detrusor muscle of wild type (WT) and Homer1^-/- mice and by demonstrating a Homer1-mediated interaction between RyR2 and Ca_v1.2 using the split GFP technique. We report that Homer1 modulates a dynamic interaction between Ca_v1.2 and RyR2 to regulate the intensity of CICR and its dependence on membrane depolarization. These findings define the molecular basis of a "two-state" model of E-C coupling by Ca_v1.2 and RyR2. In one state, Ca_v1.2 couples to RyR2 by H1b/c, which results in reduced responsiveness to membrane depolarization, and in the other state, H1a uncouples Ca_v1.2 and RyR2 to enhance responsiveness to membrane depolarization. These novel forms of interaction between RyR2 and Ca_v1.2 have important implications for smooth and possibly cardiac muscle E-C coupling.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Materials and Solutions—Fura-2/AM and Fluo-3/AM were from Teff Laboratories, Inc., Indo1 was from Molecular Probes, anti-1C subunit of Ca_v1.2 antibodies were from Alomone, and anti-RyR2 antibodies C3-33 were from Sigma. The standard bath solution A contained (in mM) 140 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 HEPES (pH 7.4 with NaOH), and 10 glucose. The standard solution B used for experiments with muscle strips and cells contained (in mM) 120.5 NaCl, 4.8 KCl, 1.2 MgSO₄, 1.2 NaH₂PO₄, 20.4 NaHCO₃, 1.6 CaCl₂, and 10 glucose, pH 7.4. High K⁺ solutions were prepared by isosmotic replacement of NaCl with KCl. The 1C, , and ₂ plasmids were generously provided by Dr. Ilya Bezprozvanny (Department of Physiology, University of Texas Southwestern Medical Center), and the RyR2 plasmid was generously provided by Dr. Andrew Marks (Department of Physiology, Columbia University).

Transfection—Human embryonic kidney (HEK)293 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were seeded in a 6-well plate at the density of 0.3 x 10⁶ cells/well on the day before transfection using the Lipofectamine and Plus reagents with 1 µg of DNA/well, in a ratio of 2:1:1:2:4 (1C: :₂: RyR2:empty vector/H1a/H1b/c).

Western Blot Analysis and Co-immunoprecipitation—Microsomes were prepared by homogenization in a buffer containing (in mM) 250 sucrose, 10 HEPES, pH 7.4, 1 EDTA, 1 dithiothreitol, and 0.2 phenylmethylsulfonyl fluoride. The homogenates were centrifuged at 1,000 x g for 10 min at 4 °C. The supernatants were collected and centrifuged at 40,000 x g for 20 min. The pellets were suspended in lysis buffer composed of (in mM) 50 Tris (pH 6.8 with HCl), 150 NaCl, 2 EDTA, 2 EGTA, and 1% Triton X-100 supplemented with protease inhibitors (0.2 phenylmethylsulfonyl fluoride, 10 µg/µl leupeptin, 15 µg/µl aptotinin, 1 mM benzamidine) and extracted by a 1-h incubation on ice. The lysate was centrifuged to remove insoluble material and analyzed by SDS-PAGE.

For the co-immunoprecipitation experiments, 150-µl extracts prepared from transfected HEK cells, as described above, were incubated with 5 µl of anti-1C antibodies overnight at 4 °C under gentle agitation. The immune complexes were collected with 30 µl of a 1:1 goat anti-IgY antibody-conjugated microbead slurry and were incubated for an additional 2 h at 4 °C. The beads were collected, washed three times with lysis buffer, and the precipitates were probed for H1b/c.

Animals—All experiments with mice have been approved by the Animal Care and Use Committee of the University of Texas Southwestern Medical Center and adhere to National Institutes of Health guidelines.

Assay of Urinary Bladder Continence—Bio-Rad filter paper sheets (Model 583) were placed over fresh cage bedding material, and the mice were placed over the filter papers while having free access to water and food. After 2 h, the filter papers were collected and the urine spots were counted under UV light.

Preparation of Detrusor Smooth Muscle (DSM) Cells and Strips—DSM cells and strips were prepared as described previously (25, 26). In brief, the bladder was excised into solution B, and the urothelium was dissected away. DSM cells were prepared by mincing the muscle and incubation for 20 min at 37 °C in 5 ml of solution B containing 1 mg/ml papain, 1 mg/ml dithiothreitol, and 1 mg/ml bovine serum albumin. The fragments were washed twice with solution B and incubated at 37 °C for 10 min with 5 ml of solution B containing 1 mg/ml collagenase type 2, 100 µM Ca²⁺, and 1 mg/ml bovine serum albumin. The tissue was washed, and cells were released by trituration. The cells were collected by passing through 125-µm nylon mesh and were concentrated by centrifugation at 100 x g.

DSM strips were prepared by cutting the bladder muscle into three strips of 0.5 x 0.5 x 8.0 mm. The strips were stretched (x1.2 slack length), and incubated in the dark with solution B containing 10 µM Indo-1/AM or 10 µM Fura-2/AM, 0.01% pluronic F-127, and 0.02% cremophor for 4 h at room temperature. The strips were then incubated in fresh solution B for 30 min at room temperature to allow completion of dye hydrolysis and used for measurements of [Ca²⁺]_i and force (Indo-1) or [Ca²⁺]_ii only (Fura2).

Measurement of Ca_v1.2 Current in DSM Cells and HEK Cells— The whole-cell current was measured with pipette solution containing (in mM) 140 CsCl, 10 EGTA, 1 MgATP, and 10 HEPES adjusted to pH 7.2 with CsOH. The bath solution contained (in mM) 140 NaCl, 10 BaCl₂, 4 KCl, and 10 HEPES adjusted to pH 7.4 with NaOH. When desired, recombinant H1a and H1b/c prepared in MgATP-free pipette solution were diluted into the pipette solution just before use. Ca_v1.2 current was recorded using Ba²⁺ as the current carrier to avoid the [Ca²⁺]_i-mediated inhibition of the current. The current was sampled at 10 kHz and filtered at 1 kHz. Current/voltage relationships were obtained by holding the membrane potential at -80 mV and stepping at 10-mV intervals for 800 ms to +60 mV. Clampex and Clampfit software were used for data acquisition and analysis.

Measurement of [Ca²⁺]_i and Force in Smooth Muscle Strips— Ca²⁺ and force of muscle strips were measured as described before (25). In brief, Indo-1-loaded strips were mounted on a force transducer and were illuminated at 365 nm. Emitted light at 405 and 485 nm was used to obtain the 405/485 ratio. When only [Ca²⁺]_i was measured, Fura2-loaded DSM strips were taped onto glass coverslips, and fluorescence was measured by illumination at 350 and 380 nm, and the emitted light was used to calculate the 350/380 ratio.

Measurement of [Ca²⁺]_i in HEK Cells—Thirty hours after transfection, the HEK cells were trypsinized and replated onto glass coverslips. After 18 h, the cells were incubated in solution A containing 10 µM Fura-2/AM for 30 min at room temperature. Fluorescence was recorded at an excitation wavelength of 350 and 380 nm, and the emitted light at 500 nm was used to calculate the 350/380 ratio.

Preparation of Split GFP-tagged Ca_v1.2 and RyR2—The 1C was tagged with the C-terminal half of GFP as follows. First, the stop codon of 1C was removed by PCR amplification using the primers 5'-GGGGTACCATGGTCAATGAAAACACGAG-3' (sense; the underlined region is a KpnI site) and 5'-ATGCGGCCGCTCAGGTTGCTGACATAGGAC-3' (antisense; the underlined region is a NotI site). The bolded base is an insert needed to make the downstream half GFP in-frame with 1C. The PCR product of 1C was cloned into pcDNA3.1(+). Positive clones were confirmed by sequencing and referred as 1CS. Thereafter, the C-terminal half of GFP (amino acids 158-237) was amplified using the primers 5'-ATGCGGCCGCGGCGGCAGCGGCAGCGGCAAGAATGGAATCAAAGTTAA-3' (sense; underlined region is a NotI site, and bolded region is a linker) and 5'-ATGCGGCCGCCTATTTGTATAGTTCATCCA (antisense; underlined region is a NotI site) and inserted into the NotI site of the C-terminal of 1CS. Positive clones were confirmed by sequencing and by current measurement.

The second half GFP was fused to the N terminus of RyR2 as follows. The N-terminal half GFP (amino acids 1-157) was amplified by the sense primer 5'-ATAGCGGCCGCATGAGTAAAGGAGAAGAAC-3' and antisense primer 5'-ATGCGGCCGCTGCCGCTGCCGCCTTGTTTGTCTGCCATGATGTAGAC-3' (the underlined region is a NotI site, and the bolded region is a linker) and inserted into the NotI site of the RyR2 plasmid, which is located upstream of the open reading frame of RyR2. Positive clones were confirmed by sequencing and caffeine-mediated Ca²⁺ release.

Measurement of GFP Fluorescence and [Ca²⁺]_i by Confocal Microscopy—Images were acquired with a Bio-Rad 1024 laser scanning confocal microscope as single images at low rate/high resolution or by continuous monitoring at a resolution of two images/sec (see Fig. 7). For measurement of depolarization-triggered CICR, after monitoring the GFP fluorescence, the cells expressing split GFP-tagged Ca_v1.2 and RyR2 and H1b/c were loaded with Fluo-3 by incubation with Fluo-3/AM for 20 min at room temperature or until Fluo-3 fluorescence was 20-fold higher than the GFP fluorescence. GFP and Fluo-3 fluorescence was measured using the 488 line of the microscope.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Deletion of Homer1 Increases Detrusor Muscle Activity—The amino acid sequences 812-817 and 1964-1967 in RyR2 and 1526-1530 in the smooth and cardiac muscle 1C isoform of Ca_v1.2 are proline-rich, Homer binding consensus sequences, and indeed, RyR2 (18) and Ca_v1.2 (see below) bind Homer. Therefore, it was of interest to determine whether the Homers participate in E-C coupling. We followed urinary bladder function, because the detrusor muscle expresses RyR2 (21) and Ca_v1.2 (22), and detrusor muscle contraction is mediated by CICR. Fig. 1, A-C, shows measurement of bladder continence as reported by urination patterns of freely moving WT mice and mice from which the Homer1, Homer2, or Homer3 genes were deleted. WT, Homer2^-/-, and Homer3^-/- mice produced an average of 2.7 ± 0.7, 2.6 ± 0.9, and 6.5 ± 2.6 (n = 3-15, trails) respectively, large and medium urine spots in 2 h. By contrast, the Homer1^-/- mice produced 169 ± 42 (n = 18) small urine spots during the same period of time, indicating overactive bladder in the Homer1^-/- mice.

Overactive bladder can result from altered detrusor muscle activity and/or altered neuronal control of muscle function, because the L-type Ca²⁺ channels and the Ca²⁺ release channels in both cell types can bind Homer. Moreover, deletion of the K⁺ channel mSlo1 also results in overactive bladder, (27) and therefore deletion of Homer1 may affect the activity of mSlow1 or the membrane potential. We consider this unlikely, because mSlo1 does not have a Homer binding ligand. In addition, although the membrane potential in the isolated cells is low because of the extensive collagenase digestion and the trituration needed to isolate the cells, the membrane potential is similar in WT (-17 ± 3.2 mV) and Homer1^-/- cells (-16.8 ± 2.8 mV).

Because we are interested in muscle E-C coupling, we asked whether deletion of Homer1 affected muscle function independent of neuronal function. Therefore, to test for a specific role of Homer1 in muscle E-C coupling, we measured [Ca²⁺]_i and contraction in isolated, urothelium-free, detrusor muscle strips from WT and Homer1^-/- mice. Fig. 1D shows representative traces of Ca²⁺ and contraction measurements in response to stimulation with 10 µM carbachol of muscle strips from WT mice. The summary in Fig. 1, E and F, shows that deletion of Homer1 results in increased sensitivity of the muscle to stimulation of the muscarinic receptors with carbachol, whereas not affecting the [Ca²⁺]_i or force response to maximal stimulation with 10 µM carbachol. Notably, Fig. 1, G and H, shows that the same effect was observed in response to depolarizing the muscle strips with high extracellular K⁺ (K⁺_o), indicating that the effect of deletion of Homer1 does not involve specific biochemical pathways stimulated by carbachol but rather involves E-C coupling directly. Moreover, these results indicate that deletion of Homer1 specifically affected muscle function.

View larger version (42K): [in this window] [in a new window]   FIGURE 1. Detrusor muscle function in Homer1-/- mice. Urinary bladder continence was assayed by counting urine spots after placing WT (A) or Homer1-/- (B) mice for 2 h in cages lined with filter paper. C summarizes the results obtained with WT (n = 15), Homer1-/- (n = 9), Homer2-/- (n = 4), and Homer3-/- mice (n = 3). D, sample traces of simultaneous measurement of peak [Ca2+]i and force in response to the stimulation of the detrusor muscles from WT mice with 10 µM carbachol. The dose response for carbachol-stimulated [Ca2+]i (E) and force (F) was measured in detrusor muscle strips from four WT and four Homer1-/- mice. Two strips were used from each bladder. The same number of mice and strips were used to measure the muscle response to membrane depolarization by elevated K+o (G and H). Results are presented as mean ± S.E.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. RyR2 activity in WT and Homer1-/- detrusor muscle. Three detrusor muscle strips from each of three WT (A and C) and three Homer1-/- (B and C) mice were used to measure the [Ca2+]i increase in response to the indicated concentrations of caffeine. The amount of RyR2 was assayed by Western blot (D).

Homer1 Activates Ca_v1.2—An increased Ca_v1.2 activity in the Homer1^-/- mice could account for the increased sensitivity of the muscle to membrane depolarization. Measurement of Ca_v1.2-mediated whole-cell Ba²⁺ current in detrusor muscle cells revealed an unexpected 39 ± 12% (n = 8) reduction in Ca_v1.2 current (Fig. 3), opposite of what is expected from the increased sensitivity of the muscle to membrane depolarization. Ba²⁺ was used as a charge carrier to avoid Ca²⁺-mediated inactivation of the channel and exclude effect of Homer1 on regulation of the channel by Ca²⁺. In addition, Homer1 does not affect the regulation of Ca_v1.2 by voltage (Fig. 3, B and C), suggesting that Homer1 increases the current amplitude of Ca_v1.2. Fig. 3D shows that the higher Ca_v1.2 current in WT cells was not due to reduced expression of Ca_v1.2 in the Homer1^-/- cells. In fact, deletion of Homer1 slightly increased expression of Ca_v1.2 by 1.2 ± 0.07-fold (n = 4), which may be an adaptive response to the reduction in Ca_v1.2 current. These findings are similar to a recent report showing that infusion of H1a enhances Ca²⁺ influx via the L-type Ca²⁺ channel in neocortex pyramidal cells (19).

Presence of a Homer binding sequence (amino acids 1526-1530) in the 1C subunit of Ca_v1.2 raised the possibility that Homer1 binds to and directly activates Ca_v1.2. To test this possibility, we first determined binding of H1b/c to 1C alone and also to 1C+ and 1C++₂ subunits of Ca_v1.2 by a co-immunoprecipitation assay. Fig. 4A shows that H1b/c does co-immunoprecipitate with 1C, and expression of the and ₂ subunits does not reduce or enhance the binding. Next, we tested whether infusion of the dissociating H1a and the multimerizing of H1b/c into WT cells affect Ca_v1.2 current. Unlike the finding in pyramidal cells (19), infusion of either Homer isoform had no effect on Ca_v1.2 current in WT cells (Fig. 4B). By contrast, infusion of either H1a or H1b/c activated Ca_v1.2 in Homer1^-/- cells and restored the current amplitude to that measured in WT cells. Importantly, a point mutation in the EVH domain of Homer (W27A) that destroys Homer binding to proline-rich sequences in target proteins (2) completely prevented activation of Ca_v1.2 by H1a and H1b/c. To further verify activation of Ca_v1.2 by the two Homer isoforms, we measured the effect of the recombinant Homers on Ca_v1.2 expressed in HEK cells. Fig. 4D shows that H1a and H1b/c activated Ca_v1.2, and H1b/c was slightly more potent than H1a.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Cav1.2 activity in WT and Homer1-/- detrusor muscle cells. The whole-cell Ba2+ currents were measured in WT and Homer1-/- detrusor muscle cells. A shows sample traces, and B shows the voltage dependence of the current recorded from at least seven cells from each phenotype. In C, the current was normalized to the maximal current measured with cells from each of the phenotypes to show that deletion of Homer1 did not affect the voltage dependence of the current. D shows analysis of Cav1.2 expression in cells from four WT and four Homer1-/- mice.

Reconstitution of Homer1 Effect on E-C Coupling in HEK Cells— The results in Figs. 1, 2, 3, 4 raised a conundrum. On the one hand, the deletion of Homer1 enhanced E-C coupling, indicating that Homer1 should inhibit Ca_v1.2 or RyR2 or both. On the other hand, deletion of Homer1 has no effect on the activity of RyR2, and Homer1 activates Ca_v1.2. A potential solution for this conundrum is that Homer1 mediates a communication between Ca_v1.2 and RyR2 to regulate CICR and muscle contraction. To this end, we reconstituted the effect of Homer1 on E-C coupling in the expression system of HEK cells. Fig. 5A shows that expression of Ca_v1.2 and RyR2 resulted in a depolarization- and caffeine-activated [Ca²⁺]_i increase, indicating that both Ca_v1.2 and RyR2 are active. Expression of the complexes dissociating H1a increased the sensitivity, whereas expression of the complexes forming H1b/c reduced the sensitivity of membrane depolarization to increase [Ca²⁺]_i. Hence, although both H1a and H1b/c activate Ca_v1.2 (Fig. 4), they have the opposite effect on CICR initiated by activation of Ca_v1.2. Notably, the effects of H1a and H1b on CICR reproduce the finding in vivo. Thus, it is possible to reconstitute the effect of Homer1 in E-C coupling by expression of the respective Homers together with RyR2 and Ca_v1.2 in HEK cells.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Interaction and activation of Cav1. 2 by Homer1. A shows co-immunoprecipitation of the 1C subunit of Cav1.2 with H1b/c. HEK cells were transfected with myc-H1b/c and 1C alone or together with the and +2 subunits of Cav1.2. Cell extracts were used to immunoprecipitate the 1C, and the immunoprecipitated proteins were blotted for myc. The controls were transfected with H1b/c and empty vector. Isolated detrusor smooth muscle cells were prepared from WT (B) or Homer1-/- mice (C) and used to measure the whole-cell current. As indicated next to the traces, the cells were infused with 5.0µg/ml H1a or 6.3 µg/ml H1b/c through a patch pipette for 7-10 min after break-in to allow equilibration of proteins. For controls, the cells were infused with 5.5 µg/ml H1a(W27A) and 6.8 µg/ml H1b/c(W27A) for 7-10 min after break-in (not shown for WT cells). The columns show mean ± S.E. of the peak current recorded from at least six cells under each condition. In D, HEK cells were transfected with 1C + + 2 subunits of Cav1.2 and infused with H1a or H1b/c, and Cav1.2 current was recorded 7-10 min after break-in. The columns show the average from 5-9 cells under each condition. * p < 0.01.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Reconstitution of Homer1-regulated CICR in HEK cells. HEK cells transfected with Cav1.2 and RyR2 (A) and H1a (B), or H1b/c (C) were depolarized by exposure to the indicated K+o concentrations, and the increase in [Ca2+]i was measured. At the end of each experiment, the cells were exposed to 20 mM caffeine (Caff) to assess the activity of RyR2. For the summary in D, the results were normalized with respect to the response measured with 80 mM K+o, which was taken as 100% (n = 4).

View larger version (59K): [in this window] [in a new window]   FIGURE 6. Homer1-mediated interaction between Cav1. 2 and RyR2 monitored with split GFP. GFP-(1-157)-RyR2 and GFP-(158-237)-1C + + 2 were expressed in HEK cells alone (A) and with H1a (B), or H1b/c (C). The model shows the proposed effects of H1a and H1b/c on the interaction between Cav1.2 and RyR2 and shows the % of fluorescent cells under each condition (D). E, after monitoring the GFP fluorescence of cells expressing GFP-(1-157)-RyR2 + GFP-(158-237)-1C + H1b/c, the cells were loaded with Fluo 3 and depolarized with the indicated K+o to monitor activity of CICR and were then incubated with 20 mM caffeine (Caff) to measure the response to maximal activation of RyR2. Note that the response to caffeine was independent of the intensity of the response to K+o.In F, the cells were grouped as expressing low or high GFP fluorescence, and their response to K+o depolarization was calculated as the percentage of their response to 20 mM caffeine. G shows similar level of Cav1.2 and RyR2 under all conditions.

To determine the role of Homer1 in the interaction between Ca_v1.2 and RyR2, the fusion proteins were expressed in HEK cells alone or with H1a or H1b/c. Panels A-C of Fig. 6 show representative images, and panel D is the summary. The superimposed bright field and fluorescent images show that, when Ca_v1.2 and RyR2 were expressed alone, 7 ± 2% of the cells showed fluorescence. Expression of the dissociating H1a, together with the channels, almost completely eliminated the fluorescent cells. Note that Ca_v1.2, RyR2, and CICR are fully active and more sensitive to membrane depolarization in these cells (Fig. 5). In sharp contrast, expression of the multimerizing H1b/c dramatically increased the number of fluorescent cells to 74 ± 13%.

As depicted in the model, the results in Fig. 6, A-D, indicate that H1a loosens and H1b/c tightens the interaction between Ca_v1.2 and RyR2. Together with the results in Fig. 5, loosening the interaction between the channels leads to enhanced efficiency of CICR, and tightening the interaction leads to reduced efficiency of CICR. Intuitively, we expected the opposite results. That is, that enhanced interaction between the channels would lead to better access of the Ca²⁺ entering through Ca_v1.2 to the RyR2 and activation of CICR. To further verify the findings in Fig. 6, A-D, we attempted to correlate between the intensity of the GFP fluorescence and response of the cells to depolarization. In each experiment, the cells with 40% or below the maximally recorded GFP fluorescence were considered as having low fluorescence and all others as having high fluorescence. Fig. 6E displays representative traces, and Fig. 6F is the summary. Only cells with relatively low fluorescence routinely responded to depolarization with 22.5 mM K⁺_o, whereas stimulation with 80 mM K⁺_o or 20 mM caffeine similarly increased [Ca²⁺]_i in all cells. Cells with high GFP fluorescence fell into two categories, those that modestly responded to depolarization with K⁺_o and about 20% that did not respond to membrane depolarization. Most notably, caffeine increased [Ca²⁺]_i in all fluorescent cells, indicating that the channels were active in the cells that did not respond to membrane depolarization. Therefore, the lack of response to depolarization must be due to impaired E-C coupling between Ca_v1.2 and RyR2.

To date, the split GFP technique has been used only to study passive protein-protein interaction (28-30). We reasoned that, because Homer1 is the natural ligand mediating the interaction between Ca_v1.2 and RyR2 to reconstitute the GFP fluorescence, we should be able to monitor conformational changes during E-C coupling by monitoring changes in GFP fluorescence. For these experiments, HEK cells were transfected with Ca_v1.2, RyR2, and H1b/c, and GFP fluorescence was monitored at a temporal resolution of two images/s. Fig. 7 shows that membrane depolarization and stimulation with carbachol caused a rapid increase in GFP fluorescence that reversed upon the removal of the stimulus. An increase in GFP fluorescence indicates altered interaction between Ca_v1.2 and RyR2 during initiation of E-C coupling. Interestingly, the GFP fluorescence did not change in cells with very high GFP fluorescence (marked by arrows). The same cells did not respond to membrane depolarization by a [Ca²⁺]_i increase.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Unlike skeletal muscle E-C coupling that involves direct interaction between Ca_v1.1 and RyR1, smooth and cardiac muscle E-C coupling does not involve direct interaction between Ca_v1.2 and RyR2. Accordingly, all models of smooth and cardiac muscle E-C coupling assume that Ca_v1.2 and RyR2 do not interact but affect the activity of each other only through changes in Ca²⁺ at the Ca²⁺ release unit (12-14). The present work calls into question this assumption by reporting a novel mode of interaction between Ca_v1.2 and RyR2 that is mediated by Homer1. Both channels express Homer binding sequences and both bind Homer (Fig. 4) ((18)). The regulation of E-C coupling in vivo is specific to Homer1, because a change in E-C coupling and muscle contraction was observed only in Homer1^-/- mice. This is not due to selective expression of Homer1, because cardiac and smooth muscle express all Homer isoforms (31). It is more likely that the action of the Homers is pathway-specific. In this respect, we showed earlier that Homer2 (but not Homer1) accelerates the GAP activity of RGS proteins and PLC to regulate IP₃ production and Ca²⁺ signaling (10).

View larger version (60K): [in this window] [in a new window]   FIGURE 7. Dynamic interaction between Cav1. 2 and RyR2. HEK cells expressing GFP-(1-157)-RyR2 + GFP-(158-237)-1C + H1b/c (A and B) were depolarized with 80 mM K+o (A) or stimulated with 1 mM carbachol (B). The change in GFP fluorescence was monitored from cells expressing low GFP (marked by yellow regions of interest), normalized, and the averages are shown below the images. The fluorescence of cells expressing high GFP fluorescence (marked by arrows) did not change.

View larger version (26K): [in this window] [in a new window]   FIGURE 8. A model depicting the role of Homer1 isoforms in E-C coupling. H1b/c solidifies the interaction between Cav1.2 and RyR2 to tame the conformational transition of Cav1.2 and/or RyR2 to reduce the efficiency of CICR. H1a loosens the interaction between the channels to facilitate the conformational transition of Cav1.2 and/or RyR2 to increase the efficiency of CICR.

Homer1-mediated interaction of Ca_v1.2 and RyR2 is dynamic and is enhanced during the activation of E-C coupling. The enhanced interaction between the channels further indicates that Homer1 acts to negatively regulate E-C coupling. In fact, the results in Fig. 7 are the first demonstration of a dynamic change in the interaction between L-type Ca²⁺ channels and RyRs during E-C coupling. Further refinement of the split GFP technique should allow careful future analysis of this conformational change.

	FOOTNOTES

 
^* This work was supported, in part, by a grant from the American Heart Association, Inc., Texas Affiliate BG1A 0665192Y (to W. Z.), National Institutes of Health Grants DE12309 and DK38938 and the Ruth S. Harrell Professorship in Medical Research (to S. M.), by the National Institute on Drug Abuse (DA00266, DA10309), and the National Institute of Mental Health (MH068830) (to P. F. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed. E-mail: pworley{at}jhmi.edu. ² To whom correspondence may be addressed. E-mail: Shmuel.muallem{at}utsouthwestern.edu.

³ The abbreviations used are: EVH, Ena/vasodilator-stimulated phosphoprotein homology 1; E-C, excitation-contraction; CICR, Ca²⁺-induced Ca²⁺ release; RyR2, ryanodine receptor type 2; [Ca²⁺]_i, free intracellular Ca²⁺ concentration; HEK, human embryonic kidney; H1a, Homer1a; H1b/c, Homer1b/c; GFP, green fluorescent protein; TRPC, canonical transient receptor potential; IP₃, inositol 1,4,5-trisphosphate; WT, wild type; DSM, detrusor smooth muscle; IP₃R, IP₃ receptor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 

1. Szumlinski, K. K., Kalivas, P. W., and Worley, P. F. (2006) Curr. Opin. Neurobiol. 16, 251-257[CrossRef][Medline] [Order article via Infotrieve]
2. Yuan, J. P., Kiselyov, K., Shin, D. M., Chen, J., Shcheynikov, N., Kang, S. H., Dehoff, M. H., Schwarz, M. K., Seeburg, P. H., Muallem, S., and Worley, P. F. (2003) Cell 114, 777-789[CrossRef][Medline] [Order article via Infotrieve]
3. Fagni, L., Worley, P. F., and Ango, F. (2002) Science's STKE 2002 137, RE8
4. Sgambato-Faure, V., Xiong, Y., Berke, J. D., Hyman, S. E., and Strehler, E. E. (2006) Biochem. Biophys. Res. Commun. 343, 630-637[CrossRef][Medline] [Order article via Infotrieve]
5. Kiselyov, K., Shin, D. M., Luo, X., Ko, S. B., and Muallem, S. (2002) Adv. Exp. Med. Biol. 506, 175-183[Medline] [Order article via Infotrieve]
6. Bers, D. M. (2004) J. Mol. Cell. Cardiol. 37, 417-429[CrossRef][Medline] [Order article via Infotrieve]
7. Zhang, H., Fu, Y., Altier, C., Platzer, J., Surmeier, D. J., and Bezprozvanny, I. (2006) Eur. J. Neurosci. 23, 2297-2310[CrossRef][Medline] [Order article via Infotrieve]
8. Kammermeier, P. J. (2006) BMC Neurosci. 7, 1[CrossRef][Medline] [Order article via Infotrieve]
9. Sala, C., Roussignol, G., Meldolesi, J., and Fagni, L. (2005) J. Neurosci. 25, 4587-4592[Abstract/Free Full Text]
10. Shin, D. M., Dehoff, M., Luo, X., Kang, S. H., Tu, J., Nayak, S. K., Ross, E. M., Worley, P. F., and Muallem, S. (2003) J. Cell Biol. 162, 293-303[Abstract/Free Full Text]
11. Kiselyov, K., Kim, J. Y., Zeng, W., and Muallem, S. (2005) Pfluegers Arch. 451, 116-124[CrossRef][Medline] [Order article via Infotrieve]
12. Franzini-Armstrong, C. (2004) Biol. Res. 37, 507-512[Medline] [Order article via Infotrieve]
13. Wehrens, X. H., Lehnart, S. E., and Marks, A. R. (2005) Annu. Rev. Physiol. 67, 69-98[CrossRef][Medline] [Order article via Infotrieve]
14. Kotlikoff, M. I. (2003) Prog. Biophys. Mol. Biol. 83, 171-191[CrossRef][Medline] [Order article via Infotrieve]
15. Feng, W., Tu, J., Yang, T., Vernon, P. S., Allen, P. D., Worley, P. F., and Pessah, I. N. (2002) J. Biol. Chem. 277, 44722-44730[Abstract/Free Full Text]
16. Hwang, S. Y., Wei, J., Westhoff, J. H., Duncan, R. S., Ozawa, F., Volpe, P., Inokuchi, K., and Koulen, P. (2003) Cell Calcium 34, 177-184[CrossRef][Medline] [Order article via Infotrieve]
17. Ward, C. W., Feng, W., Tu, J., Pessah, I. N., Worley, P. K., and Schneider, M. F. (2004) J. Biol. Chem. 279, 5781-5787[Abstract/Free Full Text]
18. Westhoff, J. H., Hwang, S. Y., Duncan, R. S., Ozawa, F., Volpe, P., Inokuchi, K., and Koulen, P. (2003) Cell Calcium 34, 261-269[CrossRef][Medline] [Order article via Infotrieve]
19. Yamamoto, K., Sakagami, Y., Sugiura, S., Inokuchi, K., Shimohama, S., and Kato, N. (2005) Eur. J. Neurosci. 22, 1338-1348[CrossRef][Medline] [Order article via Infotrieve]
20. Wray, S., Burdyga, T., and Noble, K. (2005) Cell Calcium 38, 397-407[CrossRef][Medline] [Order article via Infotrieve]
21. Ji, G., Feldman, M. E., Greene, K. S., Sorrentino, V., Xin, H. B., and Kotlikoff, M. I. (2004) J. Gen. Physiol. 123, 377-386[Abstract/Free Full Text]
22. Wegener, J. W., Schulla, V., Lee, T. S., Koller, A., Feil, S., Feil, R., Kleppisch, T., Klugbauer, N., Moosmang, S., Welling, A., and Hofmann, F. (2004) FASEB J. 18, 1159-1161[Abstract/Free Full Text]
23. Collier, M. L., Ji, G., Wang, Y., and Kotlikoff, M. I. (2000) J. Gen. Physiol. 115, 653-662[Abstract/Free Full Text]
24. Nakai, J., Ogura, T., Protasi, F., Franzini-Armstrong, C., Allen, P. D., and Beam, K. G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1019-1022[Abstract/Free Full Text]
25. Huang, G., Yao, J., Zeng, W., Mizuno, Y., Kamm, K. E., Stull, J. T., Harding, H. P., Ron, D., and Muallem, S. (2006) J. Cell Sci. 119, 153-161[Abstract/Free Full Text]
26. Isotani, E., Zhi, G., Lau, K. S., Huang, J., Mizuno, Y., Persechini, A., Geguchadze, R., Kamm, K. E., and Stull, J. T. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 6279-6284[Abstract/Free Full Text]
27. Meredith, A. L., Thorneloe, K. S., Werner, M. E., Nelson, M. T., and Aldrich, R. W. (2004) J. Biol. Chem. 279, 36746-36752[Abstract/Free Full Text]
28. Zhang, S., Ma, C., and Chalfie, M. (2004) Cell 119, 137-144[CrossRef][Medline] [Order article via Infotrieve]
29. Cabantous, S., Terwilliger, T. C., and Waldo, G. S. (2005) Nat. Biotechnol. 23, 102-107[CrossRef][Medline] [Order article via Infotrieve]
30. Ozawa, T., Nishitani, K., Sako, Y., and Umezawa, Y. (2005) Nucleic Acids Res. 33, e34[Abstract/Free Full Text]
31. Soloviev, M. M., Ciruela, F., Chan, W. Y., and McIlhinney, R. A. (2000) Eur. J. Biochem. 267, 634-639[Medline] [Order article via Infotrieve]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				N. Chopra, T. Yang, P. Asghari, E. D. Moore, S. Huke, B. Akin, R. A. Cattolica, C. F. Perez, T. Hlaing, B. E. C. Knollmann-Ritschel, et al. Ablation of triadin causes loss of cardiac Ca2+ release units, impaired excitation-contraction coupling, and cardiac arrhythmias PNAS, May 5, 2009; 106(18): 7636 - 7641. [Abstract] [Full Text] [PDF]

				J. A. Stiber, Z.-S. Zhang, J. Burch, J. P. Eu, S. Zhang, G. A. Truskey, M. Seth, N. Yamaguchi, G. Meissner, R. Shah, et al. Mice Lacking Homer 1 Exhibit a Skeletal Myopathy Characterized by Abnormal Transient Receptor Potential Channel Activity Mol. Cell. Biol., April 15, 2008; 28(8): 2637 - 2647. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 282/19/14283    most recent M611529200v2 M611529200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Huang, G. Articles by Zeng, W. Search for Related Content PubMed PubMed Citation Articles by Huang, G. Articles by Zeng, W. Social Bookmarking                      What's this?