©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of Dihydropyridine Receptor II-III Loop Peptide Regulates Skeletal Muscle Calcium Release Channel Function
EVIDENCE FOR AN ESSENTIAL ROLE OF THE beta-OH GROUP OF Ser(*)

(Received for publication, February 14, 1995; and in revised form, May 5, 1995)

Xiangyang Lu Le Xu Gerhard Meissner (§)

From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

In vertebrate skeletal muscle, excitation-contraction coupling may occur by a mechanical coupling mechanism involving protein-protein interactions between the dihydropyridine receptor (DHPR) of the transverse tubule membrane and the ryanodine receptor (RYR)/Ca release channel of the sarcoplasmic reticulum membrane. We have previously shown that the cytoplasmic II-III loop peptides of the skeletal and cardiac muscle DHPR alpha1 subunits (SDCL and CDCL, respectively) activate the skeletal muscle RYR. We now report that cyclic AMP-dependent protein kinase-mediated phosphorylation of Ser of SDCL yields a peptide that fails to activate the RYR, as determined in [^3H]ryanodine binding and single channel measurements. The phosphorylated SDCL bound to the skeletal muscle but not cardiac muscle RYR, and the binding could be displaced by the unphosphorylated SDCL. A mutant SDCL with a Ser Ala substitution failed to activate the RYR, but was still able to bind. Similarly, a Ser Ala substitution in CDCL yielded a peptide that failed to activate the skeletal RYR. Use of three smaller overlapping peptides within the SDCL region identified an amino acid region from 666 to 726 including Ser, which bound to and activated the skeletal muscle RYR. These results suggest that cyclic AMP-dependent protein kinase-mediated phosphorylation of the DHPR alpha(1) subunit may play a role in the functional interaction of the DHPR and RYR in skeletal muscle.


INTRODUCTION

In skeletal and cardiac muscle excitation-contraction (E-C) (^1)coupling, a muscle action potential activates the Ca release channel in an intracellular Ca storing membrane compartment, the sarcoplasmic reticulum (SR). The SR Ca release channels are also known as ryanodine receptors (RYR) because of their ability to bind the plant alkaloid ryanodine with high affinity and specificity(1, 2) . In vertebrate skeletal muscle, the SR Ca release channels are thought to be linked to another Ca channel (L-type), also known as the dihydropyridine receptor (DHPR), which is located in infoldings of the surface membrane, the transverse (T-) tubule(3) .

Different E-C coupling mechanisms exist in vertebrate skeletal and cardiac muscle. A major difference is that E-C coupling in cardiac muscle is dependent on extracellular Ca, whereas skeletal muscle E-C coupling is not. In cardiac muscle, the entry of extracellular Ca via a voltage-sensitive DHPR/Ca channel is required to trigger Ca release from SR(4) . In contrast, in vertebrate skeletal E-C coupling, the Ca release channels are thought to be regulated by a skeletal muscle DHPR isoform through protein-protein interactions(5, 6) . In addition to a direct activation by the DHPR, an involvement of Ca in activating the SR Ca release channel during E-C coupling has been described(6) .

The skeletal and cardiac muscle DHPRs are related oligomeric protein complexes, which comprise up to five subunits, all of which have cytoplasmic domains that could potentially interact with the SR Ca release channel(7) . Similarly, skeletal and cardiac muscle express two structurally related Ca release channels composed of four identical 565-kDa polypeptides(1, 2) . In addition to the DHPR, several endogenous effector molecules were shown to regulate the RYR. These include small diffusible molecules such as Ca, Mg, and ATP, and proteins such as calmodulin, FK506-binding protein, and triadin(2) . An involvement of triadin in mediating the functional interaction between the skeletal muscle DHPR and RYR has been suggested(8) .

Studies with skeletal and cardiac muscle chimeric cDNAs suggest that the putative II-III cytoplasmic loop region of the DHPR alpha1 subunit is a major determinant of the type of E-C coupling (skeletal or cardiac) that occurs in muscle(9) . We expressed the II-III loop regions of the skeletal and cardiac DHPR alpha1 subunits and showed that both peptides activate the purified skeletal but not the cardiac muscle Ca release channel(10) . Accordingly, the interaction was specific with respect to the skeletal muscle RYR but not with respect to the peptides.

The skeletal muscle DHPR contains several consensus phosphorylation sites including a serine residue, Ser, in the II-III loop region of the alpha1 subunit which is rapidly phosphorylated by cyclic AMP-dependent protein kinase (PKA)(11, 12) . Phosphorylation of the skeletal muscle DHPR was shown to modulate L-type Ca channel activity(13, 14) . Here, we report that phosphorylation of Ser of SDCL by PKA in vitro resulted in the formation of a peptide that failed to activate the RYR. This result provides novel evidence for a role of DHPR phosphorylation in regulating DHPR protein-RYR protein interactions in skeletal muscle.


EXPERIMENTAL PROCEDURES

Materials

Catalytic subunit of cyclic AMP-dependent protein kinase from bovine heart was purchased from Sigma and [^3H]ryanodine (54.7 Ci/mmol) from DuPont NEN. Unlabeled ryanodine was obtained from AgriSystems International (Wind Gap, PA), Pefabloc (a protease inhibitor) from Boehringer Mannheim, and phospholipids from Avanti (Birmingham, AL). All other chemicals were of analytical grade.

Site-directed Mutagenesis

The site-directed mutants SDCLS687A and CDCLS813A were generated by polymerase chain reaction (PCR). Mutagenic primers, 5`-GGA GAC CCC TGG CCA TCT TCC TGC-3` and 5`-CTT CTC CGG AGC GGC AGT CCT GGC CAG C-3`, were designed to change Ser to Ala of SDCL (15) and Ser to Ala of CDCL(16) , respectively. The megaprimers were amplified with the mutagenic primers and 5` primers in the first round PCR. The PCR products were agarose gel-purified and used as megaprimers with the 3` primers to amplify mutant SDCL and CDCL in the second round PCR. The PCR products were subcloned into the expression vector pET-11d and the mutant SDCLS687A and CDCLS813A plasmids were confirmed by DNA sequencing.

Preparation of DHPR-derived Peptides

SDCLf1 (Glu-Glu), SDCLf2 (Pro-Leu) and SDCLf3 (Lys-Leu) cDNAs (15) were amplified by PCR using SDCL as template and subcloned in the pET-11d vector. The constructed plasmids were confirmed by DNA sequencing. All peptides were expressed in Escherichia coli strain BL21(DE3) and purified by DEAE-Sephacel column chromatography, followed by hydroxyapatite column chromatography as described(10) .

In Vitro Phosphorylation

Catalytic subunit of cyclic AMP-dependent protein kinase (PKA) from bovine heart (Sigma) was reconstituted in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 20 mM dithiothreitol, and 50% glycerol and stored at -20 °C. Phosphorylation reaction was carried out by adding the peptides (1 µg) to kinase reaction buffer A (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 12 mM MgCl(2), 1 mM dithiothreitol) plus 5 µCi of [-P]ATP (3000 Ci/mmol, DuPont NEN) and 5 units of PKA in a volume of 30 µl. Reaction mixtures were incubated at room temperature for 30 min before the samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). The gel was fixed in 12% trichloroacetic acid, dried, and exposed to an x-ray film. The percentage of P incorporation was determined in the presence of 5-fold excess of ATP over SDCL by scintillation counting of the dried gel containing the P-labeled peptides. To prepare P-labeled SDCL as a probe for P-SDCL binding assay, SDCL (2 µg) was incubated with 50 µCi of [-P]ATP and 15 units of PKA in 30 µl of buffer A at room temperature for 30 min. The P-labeled SDCL was freed of [-P]ATP by gel filtration on a G-25 Sephadex column. To prepare the cold phosphorylated SDCL, 0.5 mg of SDCL was incubated with 1.5 mM ATP and 500 units of PKA in 1 ml of buffer A at room temperature for 1 h. Free ATP was removed by dialysis against 20 mM Hepes, pH 7.4, 50 mM NaCl. The phosphorylated SDCL was concentrated with a Centricon 10 concentrator (Amicon, Beverly, MA), frozen in liquid nitrogen, and stored at -80 °C.

Isolation of SR Vesicles and Purification of RYR

Heavy SR vesicles were prepared in the presence of protease inhibitors from rabbit skeletal and canine heart muscle as described(17, 18) . The RYR was purified in the presence of CHAPS as a 30 S protein complex by sucrose gradient rate centrifugation and reconstituted into proteoliposomes by removal of the detergent by dialysis(19) .

[^3H]Ryanodine Binding

Unless otherwise indicated, [^3H]ryanodine binding was determined by incubating skeletal muscle SR vesicles at 12 °C for 20 h in 100 µl of 20 mM Na-Hepes, pH 7.4, 100 mM NaCl, 0.1 mM EGTA, 0.125 mM CaCl(2), 5 mM AMP, 100 µM leupeptin, 0.5 mM Pefabloc, and 5 nM [^3H]ryanodine. Bound and free [^3H]ryanodine were determined by a filtration assay as described(19) . Nonspecific binding was determined in the presence of a 1,000-fold excess of unlabeled ryanodine.

Single Channel Recordings

Single channel measurements were performed by fusing reconstituted proteoliposomes containing the purified skeletal muscle RYR Ca release channel (19) with Muller-Rudin type planar lipid bilayers containing phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine in the ratio of 5:3:2, respectively (25 mg/ml phospholipid in n-decane)(10) . Channels were initially recorded in a symmetric KCl buffer solution containing 20 mM K-Pipes, pH 7.0, 0.25 M KCl, 0.15 mM CaCl(2), and 0.1 mM EGTA. Before the addition of the peptides, channel open probability (P(o)) was lowered to < 0.1 by the addition of EGTA to the cis chamber. The data files were acquired and analyzed using a commercially available software package (pClamp 6.0.1, Axon Instruments, Burlingame, CA) in the continuous fetchex mode, using a filter frequency setting at 2 kHz and sampling rate at 10 kHz. Channel open probabilities were determined by 50% threshold analysis as described previously(20) .

P-SDCL Overlay

Proteoliposomes containing the purified skeletal RYR and skeletal and cardiac muscle SR vesicles were dot-blotted on nitrocellulose membranes. The membranes were blocked by incubation for 1 h with 5% nonfat dry milk in binding buffer B (10 mM Hepes, pH 7.4, 50 mM NaCl, 0.1 mM EGTA, and 0.125 mM CaCl(2)). The membranes were washed once with buffer B and placed on a piece of parafilm. The binding reaction was carried out by overlaying the membranes with buffer B supplemented with P-labeled SDCL (5 10^6 cpm/ml), 1% bovine serum albumin (BSA), 100 µM leupeptin, and 0.5 mM Pefabloc. After incubation for 12 h at room temperature, the membranes were washed twice for 5 min with ice-cold buffer B, air-dried, and exposed to an x-ray film. Bound P-labeled SDCL was quantitated by scintillation counting.


RESULTS

Phosphorylation of SDCL

In purified skeletal muscle DHPR preparations, cAMP-dependent protein kinase rapidly phosphorylates Ser in the cytoplasmic II-III loop region of the DHPR alpha1 subunit (SDCL)(11, 12) . In the present study, the effects of Ser phosphorylation on SR Ca release channel function were examined, using the phosphorylated and unphosphorylated SDCL as probes in [^3H]ryanodine binding and single channel measurements. In some experiments, the cytoplasmic II-III loop peptide of the cardiac DHPR alpha1 subunit (CDCL) served as a control, as CDCL lacks a phosphorylation site corresponding to Ser in SDCL. SDCL and CDCL were expressed in E. coli, and purified by DEAE-Sephacel and hydroxyapatite column chromatography(10) . SDS-PAGE indicated that the two peptides were obtained with a purity > 99% (Fig. 1A, lanes1 and 2). The purified peptides were subjected to in vitro phosphorylation by PKA as described under ``Experimental Procedures'' and analyzed by SDS-PAGE and autoradiography. In the autoradiographs, a prominent P-labeled band with a mobility corresponding to that of SDCL was observed (Fig. 1B, lane2). A corresponding P-labeled band was not detected in the lane containing CDCL (Fig. 1B, lane1). The molar ratio of phosphate to SDCL was determined by scintillation counting to be 0.94 (data not shown), indicating only one phosphorylation site in SDCL. To confirm the presence of a single phosphorylation site in SDCL, a single point mutant with substitution of Ser to Ala was prepared. The derived peptide SDCLS687A migrated on the gels at the same position as wild type SDCL (Fig. 1A, lane3), but could no longer be phosphorylated by PKA (Fig. 1B, lane3). These results indicate that Ser is phosphorylated by PKA, and that it is the only PKA-dependent phosphorylation site in SDCL.


Figure 1: In vitro phosphorylation of purified CDCL, SDCL and SDCLS687A. A, purified peptides (2 µg of protein/lane) were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. The mobility of molecular size markers (in kDa) is indicated on the left. B, an autoradiograph of an SDS-PAGE gel shows in vitro PKA P-phosphorylation of SDCL but not of CDCL and SDCLS687A. C, alignment of amino acid sequences of SDCL (Glu to Leu) (15) and CDCL (Asp to Gln)(16) . Ser, a phosphorylation site, of SDCL, and Ser of CDCL are marked with boxes.



Effects of Phosphorylated and Mutant SDCL Peptides on [^3H]Ryanodine Binding

To assess the effects of phosphorylation on RYR function, a nonradioactively phosphorylated SDCL (SDCL-P) was prepared. In control experiments, SDCL-P did not incorporate any radioactivity when incubated with PKA and [-P]ATP. Phosphorylated peptides also showed no significant incorporation of P label following their incubation with SR vesicles under conditions comparable to the [^3H]ryanodine binding measurements. These results indicated that essentially fully phosphorylated SDCL peptides were used in the functional studies described below.

The RYR was shown to bind the neutral plant alkaloid ryanodine with nanomolar affinity in a manner that correlates well with the functional state of the Ca release channel(1, 2) . An increase in [^3H]ryanodine binding affinity by micromolar concentrations of SDCL and CDCL has been described(10) . The effects of the unphosphorylated (SDCL), phosphorylated (SDCL-P), and mutant (SDCLS687A) SDCL peptides on [^3H]ryanodine binding to SR vesicles are illustrated in Fig. 2. As shown previously (10) , the unphosphorylated SDCL increased [^3H]ryanodine binding in a dose-dependent manner. At 10 µM, SDCL increased [^3H]ryanodine binding by about 50% (Fig. 2, closedcircles). In contrast, the phosphorylated SDCL was without a significant effect. A small (10%) increase in [^3H]ryanodine binding was observed at 10 µM SDCL-P (Fig. 2, triangles). The mutant peptide SDCLS687A was without an effect on [^3H]ryanodine binding to the SR vesicles (Fig. 2, opencircles).


Figure 2: Effect of peptides on [^3H]ryanodine binding to skeletal SR vesicles. SR vesicles (150 µg of protein/ml) were incubated with 5 nM [^3H]ryanodine and the indicated amounts of SDCL (closedcircles), SDCLS687A (opencircles), CDCL (closeddiamonds), CDCLS813A (opendiamonds), and phosphorylated SDCL (triangles) as described under ``Experimental Procedures.'' Values are the means ± S.D. of two or three experiments carried out in duplicate. B(max) value for [^3H]ryanodine binding to skeletal SR vesicles in the absence of peptides was 9.5 pmol/mg as determined by Scatchard analysis. The control value (100%, without added peptides) corresponded to about 10% of the B(max) value of high affinity [^3H]ryanodine binding.



The lack of activation of [^3H]ryanodine binding by the phosphorylated and mutant peptides, shown in Fig. 2, could have been due to a decrease in the binding affinity of the peptides and/or the formation of inactive peptides. To distinguish between these possibilities, we carried out binding studies (see below) and determined the effects of the peptides on [^3H]ryanodine binding in the presence of SDCL and CDCL. Fig. 3A shows that 5 µM SDCL increased [^3H]ryanodine binding to the skeletal SR vesicles by about 50%, and the addition of the phosphorylated and mutant peptides resulted in a decrease of [^3H]ryanodine binding close to control levels (-SDCL). Half-maximal inhibition of the SDCL-activated binding activity by 5 µM SDCL-P (Fig. 3A, triangles) and 4 µM SDCLS687A (opencircles) suggested that the three peptides bound to the RYR with similar affinity.


Figure 3: Effects of SDCL-P, SDCLS687A, and CDCLS813A on [^3H]ryanodine binding to skeletal SR vesicles in the presence of SDCL or CDCL. Skeletal SR vesicles (150 µg/ml) were incubated in reaction mixture containing 5 µM SDCL (A) or 5 µM CDCL (B) and 5 nM [^3H]ryanodine in the presence of the indicated amounts of SDCLS687A (opencircles), CDCLS813A (closedcircles), and SDCL-P (triangles). Values are the means ± S.D. of two or three experiments carried out in duplicate. The control value (100%, without added peptides) corresponded to about 10% of the B(max) value of high affinity [^3H]ryanodine binding.



Effects of Mutant CDCL Peptide on [^3H]Ryanodine Binding

We were surprised to find that our mutant SDCL with Ser Ala substitution failed to activate the skeletal muscle RYR, because a Ser Ala mutation is in general considered to be a conserved mutation. This result suggested that Ser may be essential for RYR activation. To confirm the role of Ser in the formation of an active SDCL peptide, we prepared a mutant CDCLS813A with a Ser Ala substitution. We choose to replace Ser of CDCL because of its close position to Ser of SDCL in the amino acid sequence alignments (Fig. 1C). The mutant SDCLS813A was overproduced in E. coli and purified to homogeneity as described in ``Experimental Procedures'' (data not shown). Fig. 2shows that, in agreement with a previous study(10) , wild-type CDCL is more potent than SDCL in increasing [^3H]ryanodine binding to the SR vesicles. CDCL increased [^3H]ryanodine binding by about 75% at 10 µM concentration (Fig. 2, closeddiamonds). In contrast, a single point mutation in CDCL (CDCLS813A) yielded a peptide that was without an effect on [^3H]ryanodine binding (Fig. 2, opendiamonds).

The mutant CDCL peptide was as effective as the phosphorylated and mutant SDCL peptides in inhibiting the SDCL-activated RYR. Fig. 3A (closedcircles) shows that the addition of 20 µM CDCLS813A to incubation media containing 5 µM SDCL resulted in a [^3H]ryanodine binding level close to the control level (-SDCL). Similarly, the mutant SDCL and CDCL peptides were able to inhibit the CDCL-activated RYR (Fig. 3B). The addition of 5 µM CDCL increased [^3H]ryanodine binding by about 65%. This increase was nearly fully prevented by the addition of an excess (20 µM) of the mutant SDCL and CDCL peptides.

Previously, we showed that BSA activated the RYR by a mechanism different from that of the DHPR loop peptides(10) . In agreement with this observation, the mutant peptides SDCLS687A and CDCLS813A did not inhibit the increased [^3H]ryanodine binding activity of the RYR observed in the presence of BSA (data not shown).

Effects of Phosphorylated and Mutant SDCL Peptides on Single Channel Activities

An antagonistic action of the phosphorylated and mutant SDCL peptides was also observed in single Ca release channel measurements. Single, purified channels were recorded in symmetric 250 mM KCl medium. With K as the current carrier, single channel conductance was 770 picosiemens (not shown). To optimize stimulation of channel activity by SDCL, we limited the initial channel open probabilities (P(o)) to <0.1 by adjusting the free Ca concentration in the cis chamber to about 5 µM by addition of EGTA (Fig. 4, toppanels). In Fig. 4A, the addition of 100 nM SDCL increased P(o) from 0.029 to 0.121 (middlepanel). The bottomtrace of Fig. 4A shows that the subsequent addition of 100 nM phosphorylated SDCL reduced channel activity about 2-fold (P(o) = 0.062) corresponding to a level about twice that before the addition of SDCL. A comparable activation of channel activity by 100 nM SDCL and about 2-fold inhibition of the SDCL-activated channel by 200 nM SDCLS687A are shown in Fig. 4B. An inhibition of the SDCL-activated channels by SDCL-P and SDCLS687A was observed in 8 (out of 8) and 9 (out of 9) experiments, respectively (Table 1). The single channel data of Table 1are in good agreement with the [^3H]ryanodine binding measurements. Furthermore, because of the use of purified RYRs, the single channel measurements suggest that the phosphorylated SDCL and SDCLS687A antagonized the action of SDCL by a direct interaction with the RYR.


Figure 4: Effects of SDCL-P and SDCLS687A on single channel activity of the purified skeletal Ca release channel in the presence of SDCL. A, single-channel currents, shown as upward deflections, were recorded in symmetrical 0.25 M KCl media containing 8.8 µM free Cacis and 50 µM free Catrans. Holding potential = 30 mV. Toptrace, control (P(o) = 0.029); middletrace, after addition of 100 nM SDCL cis (P(o) = 0.121); bottomtrace, after the further addition of 100 nM SDCL-P cis (P(o) = 0.062). B, single channel currents, shown as upward deflections, were recorded in symmetrical 0.25 M KCl media containing 8.8 µM free Cacis and 50 µM free Catrans. Holding potential = 35 mV. Toptrace, control (P(o) = 0.025); middletrace, after addition of 100 nM SDCL cis (P(o) = 0.103); bottomtrace, after the further addition of 200 nM SDCLS687A cis (P(o) = 0.059).





Binding of P-labeled SDCL to Skeletal RYR

The phosphorylated SDCL inhibited activation of the RYR by SDCL, suggesting that SDCL-P bound to the RYR. To examine their binding to the skeletal muscle and cardiac muscle RYRs, we developed a protein overlay assay using P-labeled SDCL as a probe. Skeletal muscle and cardiac muscle SR vesicles containing a similar number of high affinity [^3H]ryanodine binding sites were dot-blotted on a nitrocellulose membrane and overlaid with P-labeled SDCL. In the autoradiographs, skeletal muscle SR vesicles, but not cardiac SR vesicles, retained P-labeled SDCL (Fig. 5), suggesting that P-labeled SDCL binds specifically to the skeletal muscle RYR. Fig. 5also shows that P-labeled SDCL binds to the purified skeletal RYR, suggesting a direct interaction between the RYR and P-labeled SDCL.


Figure 5: Specific binding of P-labeled SDCL to the skeletal RYR. A nitrocellulose membrane was dot-blotted with proteoliposomes containing the purified rabbit skeletal muscle RYR (RYR(s), 2 µg), rabbit skeletal muscle SR vesicles (SR(s), 30 µg), and canine cardiac muscle SR vesicles (SR(c), 30 µg) and overlaid with 5 10^6 cpm/ml P-labeled SDCL as described under ``Experimental Procedures.''



The interaction of RYR with other DHPR peptides was further examined in competition binding experiments. Fig. 6shows that the unphosphorylated, nonradioactively labeled phosphorylated and mutant SDCLs, and CDCL decreased binding of the P-labeled SDCL to the purified skeletal muscle RYR by 73%, 52%, 61%, and 53%, respectively (lanes 2-4 and 8). These data are consistent with binding of the peptides to the same site(s) of the skeletal RYR. However, because of difficulties in quantitating the binding data over a sufficient range of peptide concentrations, binding to different sites cannot be ruled out at this time.


Figure 6: Effects of peptides on P-labeled SDCL binding to skeletal RYR. Nitrocellulose membranes were dot-blotted with the reconstituted skeletal muscle RYR (0.8 µg) and overlaid with 5 10^6 cpm/ml P-labeled SDCL in the absence and presence of 20-30 µM each of SDCL, SDCL-P, SDCLS687A, SDCLf1, SDCLf2, SDCLf3, and CDCL as described under ``Experimental Procedures.'' Toppanel, autoradiographs of RYR overlaid with P-labeled SDCL in the absence and presence of peptides. Bottompanel, binding of P-labeled SDCL to RYR. Values were corrected for background binding (without RYR). Data are the means ± S.D. of two experiments carried out in triplicate.



Identification of Sequences within SDCL Important for Activation of the RYR

Three smaller overlapping peptides were prepared to better define the region of SDCL that activates the RYR (Fig. 7, toppanel). The bottompanel of Fig. 7shows that the N-terminal portion (SDCLf1, 61 amino acid residues) was as effective as SDCL (126 amino acids) in increasing [^3H]ryanodine binding. The middle portion (SDCLf2) was less effective and the C-terminal portion (SDCLf3) was without a significant effect. Binding studies showed that SDCLf1 decreased P-labeled SDCL binding to the RYR, in contrast to SDCLf2 and SDCLf3, which were largely ineffective in inhibiting the binding of the P-labeled peptide (Fig. 6, lanes5-7). These results indicated that a small cytoplasmic region of the skeletal muscle DHPR alpha1 subunit ranging from Glu to Glu including Ser specifically interacts with the skeletal muscle RYR.


Figure 7: Effects of SDCL, SDCLf1, SDCLf2, and SDCLf3 on [^3H]ryanodine binding to skeletal SR vesicles. Toppanel shows amino acid regions of SDCL, SDCLf1, SDCLf2 and SDCLf3. In bottompanel, SR vesicles (150 µg of protein/ml) were incubated with 5 nM [^3H]ryanodine and 10 µM each of SDCL, SDCLf1, SDCLf2, or SDCLf3. Data are the means ± S.D. of four experiments carried out in duplicate. Control value (100%, without peptides) corresponded to about 10% of the B(max) value of high affinity [^3H]ryanodine binding.




DISCUSSION

Previous studies have shown that two of the five subunits (alpha1, beta) of the skeletal muscle L-type Ca channel (DHPR) have multiple phosphorylation sites for various protein kinases including cAMP-dependent protein kinase(21) . Phosphorylation altered L-type Ca channel activity in Ca flux (13, 22) and single channel (14) measurements, and a large, voltage- and frequency- dependent potentiation of L-type Ca channel activity was described in skeletal muscle, which was due to phosphorylation by cAMP-dependent protein kinase(23) . The relevancy of these observations regarding the mechanism of E-C coupling is difficult to assess, because during normal skeletal muscle activity, the SR Ca release channel is thought to be opened via a direct physical interaction with the DHPR, which acts as a voltage-sensing molecule and not as a Ca channel(5, 6) . However, morphological evidence (3) and ligand binding measurements (24) have suggested that in skeletal muscle only a subgroup of RYRs may be directly linked to DHPRs. This observation has raised the possibility that Ca released by DHPR-linked Ca release channels could serve to amplify SR Ca release by activating Ca release channels not linked to DHPRs. An increase of DHPR/Ca channel activity by protein phosphorylation could potentiate Ca-dependent SR Ca release. In the present study we used a DHPR-derived peptide shown previously to activate the RYR, to provide novel evidence for an effect of DHPR phosphorylation on the function of DHPR-linked Ca release channels.

The basal level of phosphorylation of the skeletal muscle DHPR alpha1 subunit has been estimated by in vitro back-phosphorylation to be 35-40%. This level could be increased to 83-86% by increasing the intracellular cAMP concentration(25) . Much of this activity in the predominant (truncated) form of the DHPR alpha1 subunit could be localized to two major phosphopeptides, which both contained Ser(12) . A rapid in vitro PKA-mediated phosphorylation of Ser also indicated that this phosphorylation site may have an in vivo function. The present study confirms that PKA phosphorylates Ser and furthermore shows that phosphorylation of Ser results in loss of activation of the RYR by SDCL. This loss of activation appears to be due to the formation of an inactive peptide and not a decrease in binding affinity.

A Ser Ala substitution is a highly conserved mutation and, in general, is not considered to greatly affect the structure of the unphosphorylated protein form. However, replacement of Ser of SDCL by Ala yielded a peptide incapable of activating the RYR. This result suggests that the beta-OH of Ser is essential to form an active SDCL peptide. Mutagenesis studies with the cardiac loop peptide supported this conclusion. Although there is no PKA-phosphorylatable Ser in CDCL, amino acid sequence alignment of SDCL and CDCL reveals a Ser of CDCL close to Ser of SDCL. A single point mutation with Ser Ala substitution completely abolished the activating effect of CDCL, suggesting that Ser is also essential for the formation of an active cardiac loop peptide. Our results indicate that beta-OH of Ser of SDCL plays a crucial role in the interaction of the skeletal loop peptide with the RYR. Phosphorylation of beta-OH group of serine or its replacement by a -H will alter this interaction, resulting in an inactive peptide.

Previously, we showed that the II-III loop peptide of the cardiac DHPR alpha1 subunit (CDCL) activated the skeletal muscle RYR(10) . This result was at variance with expression studies showing that DHPR alpha1 subunit chimeras containing the skeletal muscle II-III loop conferred skeletal muscle E-C coupling, whereas chimeras containing the cardiac II-III loop showed cardiac E-C coupling(9) . Activation of the skeletal muscle RYR by both SDCL and CDCL raised the question of the specificity of this activation in our in vitro studies. This study presents three lines of evidence that favor a specific binding interaction. First, binding studies showed that both SDCL and CDCL inhibited the binding of P-labeled SDCL to the RYR. Second, phosphorylation of SDCL resulted in formation of an inactive peptide without loss of binding to the RYR. Third, a 61-amino acid sequence of SDCL was sufficient to activate the skeletal muscle RYR.

In conclusion, the data from this study demonstrate that a peptide covering a stretch of 61 amino acids in the II-III cytoplasmic loop region of the skeletal muscle DHPR alpha1 subunit activates the skeletal muscle RYR in vitro. A phosphorylation site in this sequence was found to play a crucial role in regulating the RYR in vitro. These results suggest that phosphorylation of Ser by PKA may be relevant to the function of the SR Ca release channel in skeletal muscle E-C coupling. The absence of a corresponding phosphorylation site in the II-III loop peptide of the cardiac DHPR alpha1 subunit suggests that this regulatory mechanism may be specific to skeletal muscle.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AR18687 and HL27430. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 919-966-5021; Fax: 919-966-2852.

^1
The abbreviations used are: E-C, excitation-contraction; RYR, ryanodine receptor; DHPR, dihydropyridine receptor; SR, sarcoplasmic reticulum; PKA, cyclic AMP-dependent protein kinase; SDCL, the putative cytoplasmic loop between transmembrane repeat II and III of the skeletal muscle DHPR alpha1 subunit; CDCL, the cardiac muscle isoform of SDCL; SDCLS687A, site-directed mutant SDCL with Ser Ala substitution; CDCLS813A, site-directed mutant CDCL with Ser Ala substitution; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BSA, bovine serum albumin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid.


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