Homer Protein Increases Activation of Ca2+ Sparks in Permeabilized Skeletal Muscle*

Members of the Homer family of proteins are known to form multimeric complexes capable of cross-linking plasma membrane channels (e.g. metabotropic glutamate receptor) and intracellular Ca2+ release channels (e.g. inositol trisphosphate receptor) in neurons, which potentiates Ca2+ release. Recent work has demonstrated direct interaction of Homer proteins with type 1 and type 2 ryanodine receptor (RyR) isoforms. Moreover, Homer proteins have been shown to modulate RyR-dependent Ca2+ release in isolated channels as well as in whole cell preparations. We now show that long and short forms of Homer H1 (H1c and H1-EVH1) are potent activators of Ca2+ release via RyR in skeletal muscle fibers (e.g. Ca2+ sparks) and potent modulators of ryanodine binding to membranes enriched with RyR, with H1c being significantly more potent than H1-EVH1. Homer did not significantly alter the spatio-temporal properties of the sparks, demonstrating that Homer increases the rate of opening of RyRs, with no change in the overall RyR channel open time and amount of Ca2+ released during a spark. No changes in Ca2+ spark frequency or properties were observed using a full-length H1c with mutation in the EVH1 binding domain (H1c-G89N). One novel finding with each Homer agonist (H1c and H1-EVH1) was that in combination their actions on [3H]ryanodine binding was additive, an effect also observed for these Homer agonists in the Ca2+ spark studies. Finally, in Ca2+ spark studies, excess H1c-G89N prevented the effects of H1c in a dominant negative manner. Taken together our results suggest that the EVH1 domain is critical for the agonist behavior on Ca2+ sparks and ryanodine binding, and that the coiled-coil domain, present in long but not short form Homer, confers an increase in agonist potential apparently through the multimeric association of Homer ligand.

While the functional significance of Homer is yet to be elucidated, an attractive hypothesis is that Homer proteins functionally couple cell surface receptors and intracellular Ca 2ϩ release channels into junctional signaling complexes in neurons (3). Recent results demonstrating that Homer-mediated coupling between mGluRs and InsP 3 Rs is modulated by neuronal activity (1) are consistent with a functional coupling hypothesis. This modulation of Homer-mediated coupling is thought to occur via a competitive inhibition of the binding of Homer long form (Homer 1c; H1c) as a direct consequence of expression of the immediate early gene product Homer 1 "short form" (H1a). The H1a short form is composed of an identical EVH1 binding domain as H1c long form, but lacks the CC and leucine zipper region making self-multimerization unlikely (1,3,4).
Recently Homer proteins have been shown to be expressed in both skeletal and cardiac muscle (5,6), where ryanodine receptors (RyRs), not InsP 3 Rs, mediate the release of calcium from the sarcoplasmic reticulum during excitation-contraction (E-C) coupling (7,8). Based on their sequence, RyRs are predicted to bind Homer proteins (2), and recent work has demonstrated (9) direct interaction of Homer proteins with skeletal and cardiac RyR isoforms (9 -11)). Moreover, Homer proteins have been shown to modulate RyR-dependent Ca 2ϩ release in isolated channels as well as in whole cell preparations (9 -11) leading to the hypothesis that Homer may play a functional role in Ca 2ϩ signaling in striated muscle.
Ca 2ϩ sparks arise from the opening of small groups of RyRs in muscle (12). As such, sparks can provide insight into the function of native RyR Ca 2ϩ release channels within an intact triadic structure and while associated with regulatory proteins, possibly distinct from isolated RyR in artifical membranes. In this investigation, we evaluate whether exogenously applied long and short forms of Homer 1, H1c and H1a, can exert a direct effect on RyR Ca 2ϩ release channels within skeletal muscle fibers by monitoring Ca 2ϩ sparks. We demonstrate that long and short forms of Homer H1 (H1c and H1-EVH1) are potent activators of initiation of Ca 2ϩ release via RyR in skel-etal muscle fibers (e.g. Ca 2ϩ sparks) and potent modulators of ryanodine binding to membranes enriched with RyR, with H1c being significantly more potent than the same concentration of H1-EVH1 both for Ca 2ϩ sparks and Ry binding. The changes in Ca 2ϩ sparks occurred without any significant alteration in the spatio-temporal properties of the sparks, demonstrating that Homer increases the rate of opening of RyRs, with no change in the overall RyR channel open time and amount of Ca 2ϩ released during a spark. Finally, in Ca 2ϩ spark studies, excess H1c-G89N prevented the effects of H1c in a dominant negative manner. Taken together our results suggest that the EVH1 domain is critical for the agonist behavior on Ca 2ϩ sparks and ryanodine binding and that the CC domain confers an increase in agonist potential apparently through the multimeric association of Homer ligand.

MATERIALS AND METHODS
Expression, Purification, and Verification of Homer Constructs-GST fusion constructs were made by PCR amplifying Homer-H1c ORF and 1-360-bp fragment with in-frame primers with SalI and NotI sites, and inserting the PCR products into pGEX4T-2 (Amersham Biosciences). Homer1c and H1-G89N mutants were made with the QuickChange Site-directed Mutagenesis kit (Stratagene). Mena EVH1 GST was a gift from Dr. Leahy (Johns Hopkins University). GST fusion plasmids of H1c, H1-EVH1, H1c-G89N, and Mena were transformed into BL21 cells and positive clones expanded. Cells were lysed by sonication, and the lysate was added to 1 ml of glutathione-agarose (Sigma) and sequentially washed twice with 12 ml of phosphate-buffered saline/1% Triton, two times with phosphate-buffered saline, and once with elution buffer (50 mM Tris, pH 8.0). GST fusion Homer proteins then were eluted with 10 mM glutathione (Sigma) in 50 mM Tris, pH 8.0 buffer, and eluted GST-Homer fusion proteins were dialyzed against phosphate-buffered saline at 4°C overnight. Homer proteins were verified for sequence by tandem mass spectroscopy at the Molecular Structure Facility, University of California, Davis.
Imaging Local Ca 2ϩ Release in Permeabilized Muscle Fibers-Experimental procedures and data analysis were performed as previously described by this group (13). In brief, cut segments of single fibers were isolated from ileofibularis muscle of frogs (Rana pipiens). Muscles were harvested from animals following a cold induced torpor (ice slurry), rapid decapitation, and spinal cord destruction, protocols approved by the University of Maryland Institutional Animal Care and Use Committee. Single fiber segments (3-5 mm) were manually dissected in the relaxing solution containing (mM): 120, potassium glutamate; 2, MgCl 2 ; 0.1, EGTA; 5, sodium Tris maleate; pH 7.00. Cut fiber segments were mounted under stretch in a custom chamber (14) and exposed to saponin (12 g/ml ϳ30 s.) in a standard relaxing solution Fibers were then bathed in internal solution (mM; 95, cesium glutamate; 20, creatine phosphate; 4.5, sodium Tris maleate; 13.2, cesium Tris maleate; 5, glucose; 0.1, EGTA; 1, dithiothreitol; 5, total NaATP; 0.65, free Mg 2ϩ containing 0.05 fluo-3 pentapotassium salt). Estimated [Ca 2ϩ ] free was 0.1 M. To avoid the osmotic effects of chemical permeabilization 8% dextran (41,000) was added to the solution (15).
Fibers were imaged for spontaneous Ca 2ϩ release events on an Olympus IX-70 microscope (ϫ60 -1.4 NA oil or ϫ60 -1.3 NA water objective) coupled to a Bio-Rad MRC-600 laser scanning confocal system (488 nm excitation) operated in line scan mode (1 s acquisition time; 2 ms/line, 768 pixels/line). Line scan images were computer processed to automatically identify and store spark locations using a relative threshold algorithm as described by Cheng et al. (16) and analyzed as previously described by our laboratory (13,17). Identified Ca 2ϩ sparks were analyzed for frequency of occurrence (events⅐sarcomere Ϫ1 ⅐sec Ϫ1 ), and for the following spatio-temporal properties: rise time (10 -90% ⌬F/F; ms), full-duration at half-maximal ⌬F/F (FDHM; ms), full-width at halfmaximal ⌬F/F (FWHM; m) and amplitude (peak ⌬F/F).
A sequence of 30 -50 line scan images were collected 5 min after application of the control solution (internal solution) and subsequently 5 min after exchange of internal solution containing H1c, H1-EVH1, H1c point mutant G89N, or MENA (singly or in combination as described in the figure legends), or a solution change to another control internal solution (i.e. Sham condition). Spark morphometric parameters were compared using non-parametric analysis (Mann-Whitney Test on Ranks) with significance set at p Ͻ 0.05. Frequency responses between conditions were evaluated with analysis of variance with significance set at p Ͻ 0.05.
Ryanodine Binding to Junctional Sarcoplasmic Reticulum-Junctional sarcoplasmic reticulum (JSR) membranes enriched in RyR1 were prepared from skeletal muscle of New Zealand White rabbits according to the method of Saito (18). The preparations were stored in 10% sucrose, 10 mM Hepes, pH 7.4 at Ϫ80°C until needed. Equilibrium measurement of specific high affinity [ 3 H]ryanodine ([ 3 H]Ry) binding was determined according to the method of Pessah et al. (19 -21). SR vesicles (50 g of protein/ml) were incubated with or without Homer protein in assay buffer containing HEPES (20 mM) pH 7.1, KCl (250 mM), NaCl (15 mM), varied CaCl 2 , and [ 3 H]Ry for 3 h at 37°C. The reactions were quenched by filtration through GF/B glass-fiber filters and washed twice with ice-cold harvest buffer (20 mM Tris-HCl, 250 mM KCl, 15 mM NaCl, 50 M CaCl 2 , pH 7.1). Nonspecific binding was determined by incubating SR vesicles with 1000-fold excess unlabeled ryanodine.

Long Form H1c Increases the Frequency of Occurrence of
Spontaneous Ca 2ϩ Sparks-The frequency of occurrence of Ca 2ϩ sparks provides a measure of the rate of opening of the RyR Ca 2ϩ release channel or channels that initiate the Ca 2ϩ spark. Application of long form H1c to permeabilized frog skeletal muscle fibers induced a dramatic rise in the frequency of occurrence of spontaneous Ca 2ϩ sparks, indicating an increased rate of RyR activation. Fig. 1A shows 4 successive 1-s line scan images, first in a control condition and subsequently (ϳ5 min) following application of 6.25 nM of H1c. In a group of fibers (n ϭ 4) application of 6.25 nM H1c increased Ca 2ϩ spark frequency from 0.023 Ϯ 0.01 to 0.078 Ϯ 0.01 events⅐sarc Ϫ1 ⅐ sec Ϫ1 (p Ͻ 0.05). This represents a 3.4-fold increase above the baseline frequency determined within the same fibers ( Fig. 2A). An ensemble average of single identified sparks and pseudocolor surface projection for sparks recorded in control and 25 mM H1C is presented in Fig. 1B. Events were superimposed with alignment at the midpoint of the rising phase, and averaged to obtain an image representing the population average. The average sparks were essentially the same in the presence and absence of H1c. Because of the ϳ 14-fold increase in Ca 2ϩ spark frequency in the presence of H1c compared with the control condition, the vast majority of events contributing to increased spark frequency observed are likely the direct result of H1c. Furthermore, H1c-modified fibers exhibit spark events that maintain similar spatio-temporal characteristics to those of control fibers (see Fig. 2C, below).
The concentration dependence of the H1c effect on Ca 2ϩ spark occurrence is shown in Fig. 2A. Application of 5-50 nM H1c revealed a linear rise in the frequency of Ca 2ϩ spark occurrence with concentration, indicating that 50 nM is well below the dissociation constant of the RyR for H1c and that the maximal effect of H1c on spark frequency is likely to be much greater than shown in Fig. 2A. Further increasing H1c concentration (Ն100 nM) resulted in a higher level of Ca 2ϩ spark activity and increase in non-spark fluorescence making the detection and analysis of Ca 2ϩ spark properties unreliable at these high concentrations of H1c (data not shown). A "sham" condition (n ϭ 3) consisting of a solution change to an identical internal solution (0 nM H1c), resulted in a small, non-significant increase in Ca 2ϩ spark frequency thus demonstrating that any mechanical disturbance due to changing experimental conditions had a negligible effect.
The spatio-temporal properties of a spark reflect the overall duration and amount of Ca 2ϩ released by the channels that generate that Ca 2ϩ spark (22). The effect of H1c on the spatiotemporal parameters for Ca 2ϩ sparks is evaluated between the control and 6.25 nM H1c conditions within the same fibers (n ϭ 4) in Fig. 2C. Despite the robust increase in Ca 2ϩ spark frequency, no significant differences in mean values for spark 2ms) was seen between the control and H1c, respectively. Thus, although H1c does markedly increase the rate of opening of the RyR channels that initiate the sparks (i.e. frequency of Ca 2ϩ sparks), the overall duration and amount of Ca 2ϩ released in a spark does not appear to be significantly altered by the Homer protein.
Specificity and Effectiveness of Long versus Short Form Homer 1 on Eliciting Spontaneous Ca 2ϩ Spark Frequency-As reported above, the application of 5-50 nM H1c induced a robust increase in the frequency of occurrence of spontaneous Ca 2ϩ sparks. To evaluate the specificity of H1c effects on spark activity, a point mutation of H1c (H1c-G89N) with impaired ability to bind Homer ligand, but with maintained ability to multimerize, was tested (1). This mutant exhibits negligible binding to the Homer ligand domain (1,23). When applied at a 10-fold higher concentration than that of H1c, H1c-G89N failed to significantly alter Ca 2ϩ spark frequency (Fig. 3A). These results suggest that enhanced Ca 2ϩ spark activity is mediated by the interaction between the EVH1 domain of H1c and the Homer ligand domain of RyRs. The specificity of the H1c effect toward RyR shown above was further tested by preincubation of muscle fibers with the InsP 3 R inhibitor heparin (50 g/ml).
The H1c effect in heparin-treated fibers (n ϭ 2) was similar to that of control fibers (n ϭ 2; data not shown). Application of heparin in the absence of Homer protein also had no effect on spark frequency or properties (n ϭ 2; data not shown) indicating that InsP 3 R's are not responsible for the Ca 2ϩ sparks seen in frog skeletal muscle, either in the presence or absence of exogenously applied H1c.
Short form H1a is an alternatively spliced version of H1c protein composed only of the H1-EVH1 binding domain and lacking the CC domain necessary for self-multimerization. Permeabilized muscle fibers challenged either with 30 nM (n ϭ 7), 50 nM (n ϭ 2), or 150 nM (n ϭ 2) recombinant H1-EVH1 exhibited a significant concentration-dependent increase in spontaneous Ca 2ϩ spark frequency ( Fig 2B). However, 30 -150 nM H1-EVH1 was less effective in inducing an increase in spontaneous spark activation than 5-50 nM H1c. H1-EVH1 required ϳ5-fold higher concentration than H1c to elicit similar increases in Ca 2ϩ spark occurrence, and was unable to induce the highest level of activation seen with H1c in this preparation (Fig. 2B). The spatio-temporal properties of the events measured in the presence of H1-EVH1 were not significantly different from those seen in control and H1c-treated muscle fibers (Fig. 2C). The protein Mena-EVH1 (MENA; murine Ena homologue) was used to test the specificity of H1a effects reported above. Mena-EVH1 lacks a CC domain and is structurally similar to Homer EVH1, yet it binds to a distinct proline-rich sequence independent of the Homer ligand site (1). This protein at 50 nM had no effect on the frequency of occurrence of Ca 2ϩ sparks (Fig. 3).
The Potential Role of the Homer Multimerization via the CC Domain in Ca 2ϩ Spark Activation-The current results indicate that the activity of Homer 1 toward enhancing the frequency of RyR-mediated spark activity is specific to the EVH1 domain and does not require the CC domain. However the presence of the CC domain of H1c does increase effectiveness of a given concentration of Homer by ϳ5-fold (H1c versus H1-EVH1). One possible mechanism responsible for the greater activity of H1c compared with H1a could involve the multimeric nature of H1c via the CC domain. To evaluate this possibility, H1c (6.25 nM) was allowed to equilibrate (ϳ30 min) in solution together with 10-fold excess of the non-binding point mutant H1c-G89N (above) before challenging the fiber. We predict that pre-equilibration of H1c with excess H1c-G89N should promote the formation of homodimers of H1c-G89N, heterodimers of H1c/H1c-G89N and an insignificant fraction of H1c homodimers based on the stoichiometry at equilibrium (ϳ83, 16, and 1% respectively). This stoichiometry, we hypothesize, should result in a diminution in the relative efficacy of H1c if multimerization through the CC domain is essential for high efficacy. The results summarized in Fig. 4 show that 6.25 nM H1c, which forms homodimers, promoted the most robust elevation in Ca 2ϩ spark frequency. By comparison, preincubated samples containing 6.25 nM H1c ϩ excess H1c-G89N (in which H1c homodimers constitute ϳ1%) applied to fibers greatly blunted the response expected for 6.25 nM H1c alone to a level no different than the control condition, The suppression of the H1c response by excess H1c-G89N cannot be ascribed to simple competitive inhibition at the RyR Homer ligand site since H1c-G89N is a non-binding mutant (1,23), indicating that heterodimers of Hlc/Hlc-G89N are ineffective at activating Ca 2ϩ sparks. The subsequent addition of excess H1c (50 nM) to the fibers did promote a drastic increase in Ca 2ϩ spark activation (data not shown), verifying that the spark activating potential remained.
To further evaluate this hypothesis, 30 nM H1-EVH1 was preincubated with excess (210 nM) H1c-G89N and subsequently applied to a permeabilized fiber. The activity of this mixture toward enhancing Ca 2ϩ spark activity was slightly but significantly lower than that seen with an equivalent concentration of H1a alone (Fig. 4). However, H1c-G89N is clearly not as effective in suppressing the enhanced Ca 2ϩ spark frequency elicited by H1-EVH1 short-form when compared with the suppression observed with H1c-G89N ϩ H1c. Taken together these results support a role for the CC domain in the enhanced effectiveness of a given concentration of H1c compared with H1-EVH1 toward activating Ca 2ϩ sparks in this system.
In order to further test this hypothesis, 6.25 nM H1c (long form) was pre-equilibrated with 30 nM H1-EVH1 (short form), and the fiber was challenged with the mixture. This condition resulted in an additive effect between long and short forms toward enhancing Ca 2ϩ spark frequency (ϳ6-fold over control levels; Fig. 4). This level of activation is consistent with a purely additive action between H1c (ϳ3.5-fold) and H1-EVH1 (ϳ2.5-fold) when presented individually. This is consistent with each of the individual proteins being within the linear concentration range for spark activation and with no interaction between H1c and H1-EVH1 proteins.
Homer and RyR Activation Dynamics Revealed with Ryanodine Binding-In Fig. 5

Long and Short Homer Proteins Promote RyR Activation-It
is well known that RyR-dependent SR Ca 2ϩ release is modulated by endogenous ions such as Ca 2ϩ and Mg 2ϩ (16,24,25). In addition, RyR-dependent Ca 2ϩ release is also modulated by regulatory proteins such as calmodulin (26,27) and FKBP12 (28,29), which associate at distinct binding sites on the RyR (30,31). In permeabilized and thus continuously depolarized skeletal muscle fibers, DHPR voltage sensors are inactivated (32) and unable to initiate SR Ca 2ϩ release. The appearance of Ca 2ϩ sparks in this preparation thus presumably arises from ligand-dependent activation of RyRs; most likely through calcium-induced calcium release (CICR) mechanisms (8,12).
Recent reports have demonstrated that skeletal type RyR contains the proline-rich (PPXXFS) Homer binding sequence, and recent studies have demonstrated a direct binding interaction of Homer long and short forms with RyR in GST pulldown assays (10). Recent investigations have also demonstrated that Homer increases the open probability of RyR in the bilayer and also increases the CICR sensitivity in intact skeletal muscle myotubes (10); with H1c being more effective than H1-EVH1 at the same concentration.
In contrast to these findings, a recent report by Hwang et al. (9) demonstrated that Homer short and long forms differentially regulated RyR1. In this study, long form Homer (e.g. H1c; V-1L) both bound and activated RyR1 whereas Homer short form (e.g. H1-EVH1Ј V-1s) bound to RyR1 yet did not activate FIG. 4. The potential role of the Homer multimerization via the CC domain in Ca 2؉ spark activation. A, varying Homer conditions to investigate Homer interaction are shown. Spark frequency expresses as a percentage of control. All protein concentrations listed are in nanomolar concentrations. B, schematic representation of the predicted ability of the protein to bind a Homer ligand target, to dimerize into a multimeric complex, and to cross-link target proteins. A-B, H1c (6.25 nM; red bar) was allowed to equilibrate together with 10fold excess H1c-G89N (70 nM, ϳ30 min) before application to the fiber. This condition (red-black checker) is predicted to result in homodimers of the H1c-G89N, heterodimers of H1c and H1c-G89N, and an insignificant amount of H1c homodimers based on the stoichiometry at equilibration (ϳ83, 16, and 1%, respectively). This combination greatly blunted the increase in frequency seen with the H1c alone. 30 nM H1-EVH1 (yellow bar) with excess H1c-G89N (210 nM; yellow-black checker) resulted in a small but significant reduction in Ca 2ϩ spark frequency when compared with the H1-EVH1 condition. However, H1c-G89N ϩ H1-EVH1 was clearly not as effective in suppressing Ca 2ϩ spark frequency as seen with H1c-G89N ϩ H1c. H1c (6.25 nM) pre-equilibrated with H1-EVH1 (30 nM) resulted in an additive effect in Ca 2ϩ spark activation to ϳ6-fold over control levels. This level of activation is consistent with H1c (ϳ3.5-fold) and H1-EVH1 (ϳ2.5-fold) each exerting an independent effect on Ca 2ϩ spark activation. This is consistent with each individual protein within the linear concentration range for spark activation. the channel. In addition, Homer short form dose-dependently decreased the effect of Homer long form, an effect that was not evident in our experiments. Furthermore, in contrast to the marked potentiation of spark frequency observed here for both H1c (6.25 nM resulted in a ϳ4-fold increase, see Fig 2A) and H1-EVH1 (30 nM resulted in ϳ5-fold increase, see Fig. 2B), Hwang et al. (9) found only a modest 1.8-fold increase in RyR channel P open with 100 nM H1c. The disparity between this and our current data may be due to differences in the activity of recombinant Homer peptides used for the respective studies. Based on early experiments (12) our group did consider a differential regulation of Homer long and short form on Ca 2ϩ spark behavior in the permeabilized frog fiber. In extending our experiments we realized significant variability of our results among different preparations of purified Homer protein. This variability led us to verify the sequence of all protein preparations (see "Materials and Methods") prior to performing the experiments presented here. With sequence verified protein, we largely eliminated experimental variability between preparations, and have proceeded to demonstrate Homer-dependent RyR agonist behavior of both H1c and H1-EVH1 with three different methodologies: Ca 2ϩ sparks in permeabilized frog muscle and ryanodine binding to JSR fractions in the current study, and single channel recording using reconstituted RyR1 in the BLM (10).
Our present functional studies now show a direct and additive effect of H1c and H1-EVH1 on activating Ca 2ϩ sparks in permeabilized muscle fibers. In agreement with these findings we also report that H1c and H1-EVH1 modulate ryanodine binding to RyR1 in JSR in a purely additive manner. Fulllength Homer (H1c) was a more potent agonist of the RyR channel (ϳ5-fold) than H1-EVH1 both in measurements of Ca 2ϩ spark activity and in radioligand binding analysis. The similar relative effectiveness between the two different methodologies used here, as well as previous reports demonstrating differing efficacy of H1c and H1-EVH1 in the BLM (10), suggests a similar mode of action by which Homer long form and short form activate RyR, whether in the permeabilized muscle fiber or in the isolated membrane or single channel experiments.
Ca 2ϩ Spark Properties Are Not Altered by Homer: Insight into the Homer Effect-Following RyR activation, the shape of the Ca 2ϩ spark (i.e. spatio-temporal properties) is determined by the underlying RyR channel behavior (12,22). Several reports have demonstrated that alterations in the spatial and/or temporal properties of spontaneous Ca 2ϩ sparks correlate with alterations in RyR channel gating seen in planar lipid bilayer experiments (12)(13)(14)33). In this investigation, the effect of both H1c and H1-EVH1 was specific to an increase in Ca 2ϩ spark frequency, with minimal alteration in the shape of the individual Ca 2ϩ sparks (i.e. spatio-temporal properties). It is widely accepted that Ca 2ϩ sparks arise from a CRU containing a small number of RyR channels and that the stereotypic properties of Ca 2ϩ sparks most likely are the result of some level of coordinated control. Based on this assumption, the present results support the conclusion that Homer long and short forms increase Ca 2ϩ spark frequency by increasing the probability of opening of the RyR Ca 2ϩ release channel(s) that initiate the Ca 2ϩ sparks (trigger event within the CRU), without effecting the overall RyR channel opening underlying the Ca 2ϩ spark. This finding is supported by recent work by Feng et al. (10) who demonstrated that in the BLM both Homer H1c and H1-EVH1 increased single channel gating activity without altering open dwell time.
Proposed Mechanisms for the Homer Effects Seen in this Investigation-Recent reports have hypothesized several mechanisms for Homer action on RyR ranging from a mechanical linking of related Ca 2ϩ signaling proteins (34 -36) to the tethering of Ca 2ϩ signaling proteins to the skeletal muscle triad (9). Both of these mechanisms are thought possible due to the multimerization of Homer. Homer long form (H1c) differs from H1-EVH1 in that H1c contains a Ͼ240 amino acid region on the C-terminal end of the EVH1 domain. This region is predicted to form a coiled-coil tertiary structure, which is thought to promote self-assembly of the Homer monomers into multimeric complexes (3,37). The facts that an isolated single channel in BLM can be activated upon addition of H1c (9 -11) and that H1-EVH1 activates both single channels in the BLM (10) /ml), and 148 nM (2 g/ml) H1EVH1, 24 nM (1 g/ml), 48 nM (2 g/ml of Homer1c (H1c), 24 nM (1 g/ml) H1c ϩ 74 nM (1 g/ml) EVH1. Each treatment (i.e. colored bar) was significantly different from the Control (i.e. no Homer) condition. Statistical significance between selected conditions is noted. The H1c ϩ H1-EVH1 condition is significantly different from all other groups except for the 48 nM H1c. The data shown (mean Ϯ S.D.) are from 4 -6 samples in each condition. wild type (binding) and one mutated (nonbinding) EUH1 domain appears to be ineffective in Ca 2ϩ spark activation. A possible DHPR-RyR interaction secondary to cross-linking of DHPR and RyR by wild-type multimeric H1c would not be a likely explanation for our results. First, H1-EVH1 was an agonist of RyR activation and this protein is without a CC domain and cannot form complexes. Therefore, H1-EVH1 could act on either the DHPR or the RyR, but not by linking the two proteins in any manner. Second, in the present experiments, Homer was either added to JSR membrane or to a permeabilized muscle fiber. In ryanodine binding experiments, exogenous Homer promotes ryanodine binding to JSR independent of DHPR since little or no DHPR would be present in the JSR preparation. Furthermore, in permeabilized muscle fibers the sarcolemma is disrupted and the voltage sensors are thought to be in the inactivated state where DHPR-RyR interaction would not be productive (32). Therefore, any sparks that arise in control conditions are likely spontaneous in nature, independent of the DHPR, arising solely from the RyR (no InSP3 involvement).
Recent biochemical and in vivo evidence also supports the agonist behavior of Homer being primarily on RyRs. Taken together we can suppose that the effect of Homer on the RyR is to augment RyR sensitivity to CICR (10). While we did not expressly test this hypothesis, spontaneous Ca 2ϩ sparks seen here are thought to occur via CICR mechanisms. In addition, limited experiments (n ϭ 2 fibers ; data not shown) in which a Homer challenge was performed in conditions with less CICR potential (e.g. elevated Mg 2ϩ ; 1.8 mM free ) resulted in Homer having a less robust response. This is in agreement with Homer acting to indirectly promote RyR opening through CICR mechanisms (10), thus not acting as a direct agonist of RyR opening.
Potential modes of action of short and long Homer ligands can be extrapolated from other known RyR ligands and their effect on spontaneous Ca 2ϩ sparks. In the same permeabilized muscle fiber preparation used here, low concentrations (nM) of Imperatoxin (14,38) activated one RyR channel, which often promoted the brief activation of the entire CRU thus revealing a Ca 2ϩ spark. In these experiments, the IpTxA was reported to bind to a single binding site on one RyR monomer to exert its effect. In this regard, we can envision a similar behavior of the H1-EVH1 ligand, which also has one RyR ligand sequence and thus would be expected to bind to one RyR monomer within the tetramer. With this in mind we can extrapolate the action of a ligand with multiple RyR binding sites.
The H1c monomer is predicted to form multimeric complexes thus resulting in a single ligand complex with multiple binding sites. In the permeabilized fiber, it is then possible that the H1c complex binds to multiple RyR homotetramers within the CRU thus further enhancing the CICR potential based on the number of RyRs it interacts with. Alternatively, it is possible that multimeric Homer binds RyR monomers exerting its effect within the RyR homotetramer. In this regard, the DP-4 ligand promoted Ca 2ϩ spark activity by modulating intra-RyR communication (13) establishing precedence for intramolecular RyR modulation promoting Ca 2ϩ spark activation.
Independent of either an inter-or intra-RyR binding configuration of multimeric Homer ligand, a given concentration of H1c could then have the observed 5-fold higher effect (per molecule of H1c or H1-EVH1 present) on Ca 2ϩ spark frequency (or ryanodine binding) due to either a higher affinity of the multimer for RyR(s) or to the occupancy of multiple RyR sites when a single multimer binds, or to some combination of the of the two effects. While either mechanism as well as a combination of RyR binding configurations is possible, it is unlikely that the H1c complex modulates the rate of inter-RyR or inter-CRU communication as the spatio-temporal properties of the Ca 2ϩ sparks have not changed.