Deletions of any single residues in Glu40-Ser48 loop connecting a domain and the first transmembrane helix of sarcoplasmic reticulum Ca(2+)-ATPase result in almost complete inhibition of conformational transition and hydrolysis of phosphoenzyme intermediate

Possible roles of the Glu40-Ser48 loop connecting A domain and the first transmembrane helix (M1) in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) were explored by mutagenesis. Deletions of any single residues in this loop caused almost complete loss of Ca(2+)-ATPase activity, while their substitutions had no or only slight effects. Single deletions or substitutions in the adjacent N- and C-terminal regions of the loop (His32-Asn39 and Leu49-Ile54) had no or only slight effects except two specific substitutions of Asn39 found in SERCA2b in Darier's disease pedigrees. All the single deletion mutants for the Glu40-Ser48 loop and the specific Asn39 mutants formed phosphoenzyme intermediate (EP) from ATP, but their isomeric transition from ADP-sensitive EP (E1P) to ADP-insensitive EP (E2P) was almost completely or strongly inhibited. Hydrolysis of E2P formed from Pi was also dramatically slowed in these deletion mutants. On the other hand, the rates of the Ca(2+)-induced enzyme activation and subsequent E1P formation from ATP were not altered by the deletions and substitutions. The results indicate that the Glu40-Ser48 loop, with its appropriate length (but not with specific residues) and with its appropriate junction to A domain, is a critical element for the E1P to E2P transition and formation of the proper structure of E2P, therefore, most likely for the large rotational movement of A domain and resulting in its association with P and N domains. Results further suggest that the loop functions to coordinate this movement of A domain and the unique motion of M1 during the E1P to E2P transition.     

Roles of Tyr122-hydrophobic cluster and K+ binding in Ca2+ -releasing process of ADP-insensitive phosphoenzyme of sarcoplasmic reticulum Ca2+ -ATPase

Tyr(122)-hydrophobic cluster (Y122-HC) is an interaction network formed by the top part of the second transmembrane helix and the cytoplasmic actuator and phosphorylation domains of sarcoplasmic reticulum Ca(2+)-ATPase. We have previously found that Y122-HC plays critical roles in the processing of ADP-insensitive phosphoenzyme (E2P) after its formation by the isomerization from ADP-sensitive phosphoenzyme (E1PCa(2)) (Wang, G., Yamasaki, K., Daiho, T., and Suzuki, H. (2005) J. Biol. Chem. 280, 26508-26516). Here, we further explored kinetic properties of the alanine-substitution mutants of Y122-HC to examine roles of Y122-HC for Ca(2+) release process in E2P. In the steady state, the amount of E2P decreased so that of E1PCa(2) increased with increasing lumenal Ca(2+) concentration in the mutants with K(0.5) 110-320 microm at pH 7.3. These lumenal Ca(2+) affinities in E2P agreed with those estimated from the forward and lumenal Ca(2+)-induced reverse kinetics of the E1PCa(2)-E2P isomerization. K(0.5) of the wild type in the kinetics was estimated to be 1.5 mM. Thus, E2P of the mutants possesses significantly higher affinities for lumenal Ca(2+) than that of the wild type. The kinetics further indicated that the rates of lumenal Ca(2+) access and binding to the transport sites of E2P were substantially slowed by the mutations. Therefore, the proper formation of Y122-HC and resulting compactly organized structure are critical for both decreasing Ca(2+) affinity and opening the lumenal gate, thus for Ca(2+) release from E2PCa(2). Interestingly, when K(+) was omitted from the medium of the wild type, the properties of the wild type became similar to those of Y122-HC mutants. K(+) binding likely functions via producing the compactly organized structure, in this sense, similarly to Y122-HC.     

Slow transition of phosphoenzyme from ADP-sensitive to ADP-insensitive forms in solubilized Ca2+, Mg2+-ATPase of sarcoplasmic reticulum: evidence for retarded dissociation of Ca2+ from the phosphoenzyme.

Solubilized Ca²⁺, Mg²⁺-ATPase of sarcoplasmic reticulum was phosphorylated with ATP without added MgCl₂. The phosphoenzyme formed was ADP-sensitive. Ca²⁺ in the medium was chelated after phosphorylation. This induced a slow transition of the phosphoenzyme from ADP-sensitive to ADP-insensitive forms. The ADP-sensitivity was restored by subsequent addition of CaCl₂. These results showed that the transition was caused by dissociation of Ca²⁺ bound to the phosphoenzyme. Further observations indicated that, when Ca²⁺ in the medium was chelated, Ca²⁺ bound to the phosphoenzyme was dissociated much more slowly than Ca²⁺ bound to the dephosphoenzyme. This suggests a possible formation of the occluded form of the Ca²⁺-binding site in the phosphoenzyme.     

Ca2+ occlusion and gating function of Glu309 in the ADP-fluoroaluminate analog of the Ca2+-ATPase phosphoenzyme intermediate

In the absence of ATP the sarcoplasmic reticulum ATPase (SERCA) binds two Ca(2+) with high affinity. The two bound Ca(2+) rapidly undergo reverse dissociation upon addition of EGTA, but can be distinguished by isotopic exchange indicating fast exchange at a superficial site (site II), and retardation of exchange at a deeper site (site I) by occupancy of site II. Site II mutations that allow high affinity binding to site I, but only low affinity binding to site II, show that retardation of isotopic exchange requires higher Ca(2+) concentrations with the N796A mutant, and is not observed with the E309Q mutant even at millimolar Ca(2+). Fluoroaluminate forms a complex at the catalytic site yielding stable analogs of the phosphoenzyme intermediate, with properties similar to E2-P or E1-P.Ca(2). Mutational analysis indicates that Asp(351), Lys(352), Thr(353), Asp(703), Asn(706), Asp(707), Thr(625), and Lys(684) participate in stabilization of fluoroaluminate and Mg(2+) at the phosphorylation site. In the presence of fluoroaluminate and Ca(2+), ADP (or AMP-PCP) favors formation of a stable ADP.E1-P.Ca(2) analog. This produces strong occlusion of Ca(2+) bound to both sites (I and II), whereby dissociation occurs very slowly even following addition of EGTA. Occlusion by fluoraluminate and ADP is not observed with the E309Q mutant, suggesting a gating function of Glu(309) at the mouth of a binding cavity with a single path of entry. This phenomenon corresponds to the earliest step of the catalytic cycle following utilization of ATP. Experiments on limited proteolysis reveal that a long range conformational change, involving displacement of headpiece domains and transmembrane helices, plays a mechanistic role.     

Importance of transmembrane segment M1 of the sarcoplasmic reticulum Ca2+-ATPase in Ca2+ occlusion and phosphoenzyme processing

The functional consequences of a series of point mutations in transmembrane segment M1 of sarcoplasmic reticulum Ca2+-ATPase were analyzed in steady-state and transient kinetic experiments examining the partial reaction steps involved in Ca2+ interaction and phosphoenzyme turnover. Arginine or leucine substitution of Glu51, Glu55, or Glu58, located in the N-terminal third of M1, did not affect these functions. Arginine or leucine substitution of Asp59, located right at the bend of M1 seen in the crystal structure of the thapsigargin-bound form, caused a 10-fold increase of the rate of Ca2+ dissociation toward the cytoplasmic side. Mutation of Leu60 to alanine or proline and of Val62 to alanine also enhanced Ca2+ dissociation, whereas an 11-fold reduction of the rate of Ca2+ dissociation was observed upon alanine substitution of Leu65, thus providing evidence for a relation of the middle part of M1 to a gating mechanism controlling the dissociation of occluded Ca2+ from its membranous binding sites. Moreover, phosphoenzyme processing was affected by some of the latter mutations, in particular leucine substitution of Asp59, and alanine substitution of Leu65 accelerated the transition to ADP-insensitive phosphoenzyme and blocked its dephosphorylation, thus demonstrating that this part of M1, besides being important in Ca2+ interaction, furthermore, is a critical element in the long range signaling between the transmembrane domain and the cytoplasmic catalytic site.     

Ca2+ release to lumen from ADP-sensitive phosphoenzyme E1PCa2 without bound K+ of sarcoplasmic reticulum Ca2+-ATPase

During Ca(2+) transport by sarcoplasmic reticulum Ca(2+)-ATPase, the conformation change of ADP-sensitive phosphoenzyme (E1PCa(2)) to ADP-insensitive phosphoenzyme (E2PCa(2)) is followed by rapid Ca(2+) release into the lumen. Here, we find that in the absence of K(+), Ca(2+) release occurs considerably faster than E1PCa(2) to E2PCa(2) conformation change. Therefore, the lumenal Ca(2+) release pathway is open to some extent in the K(+)-free E1PCa(2) structure. The Ca(2+) affinity of this E1P is as high as that of the unphosphorylated ATPase (E1), indicating the Ca(2+) binding sites are not disrupted. Thus, bound K(+) stabilizes the E1PCa(2) structure with occluded Ca(2+), keeping the Ca(2+) pathway to the lumen closed. We found previously (Yamasaki, K., Wang, G., Daiho, T., Danko, S., and Suzuki, H. (2008) J. Biol. Chem. 283, 29144-29155) that the K(+) bound in E2P reduces the Ca(2+) affinity essential for achieving the high physiological Ca(2+) gradient and to fully open the lumenal Ca(2+) gate for rapid Ca(2+) release (E2PCa(2) → E2P + 2Ca(2+)). These findings show that bound K(+) is critical for stabilizing both E1PCa(2) and E2P structures, thereby contributing to the structural changes that efficiently couple phosphoenzyme processing and Ca(2+) handling.     

Critical role of Val-304 in conformational transitions that allow Ca2+ occlusion and phosphoenzyme turnover in the Ca2+ transport ATPase

Site-directed mutations were produced in the distal segments of the Ca(2+)-ATPase (SERCA) transmembrane region. Mutations of Arg-290 (M3-M4 loop), Lys-958, and Thr-960 (M9 - M10 loop) had minor effects on ATPase activity and Ca(2+) transport. On the other hand, Val-304 (M4) mutations to Ile, Thr, Lys, Ala, or Glu inhibited transport by 90-95% while reducing ATP hydrolysis by 83% (Ile, Thr, and Lys), 56% (Ala), or 45% (Glu). Val-304 participates in Ca(2+) coordination with its main-chain carbonyl oxygen, and this function is not expected to be altered by mutations of its side chain. In fact, despite turnover inhibition, the Ca(2+) concentration dependence of residual ATPase activity remained unchanged in Val-304 mutants. However, the rates (but not the final levels) of phosphoenzyme formation, as well the rates of its hydrolytic cleavage, were reduced in proportion to the ATPase activity. Furthermore, with the Val-304 --> Glu mutant, which retained the highest residual ATPase activity, it was possible to show that occlusion of bound Ca(2+) was also impaired, thereby explaining the stronger inhibition of Ca(2+) transport relative to ATPase activity. The effects of Val-304 mutations on phosphoenzyme turnover are attributed to interference with mechanical links that couple movements of transmembrane segments and headpiece domains. The effects of thermal activation energy on reaction rates are thereby reduced. Furthermore, inadequate occlusion of bound Ca(2+) following utilization of ATP in Val-304 side-chain mutations is attributed to inadequate stabilization of the Glu-309 side chain and consequent defect of its gating function.     

Selective inhibition by lasalocid of hydrolysis of the ADP-insensitive phosphoenzyme in the catalytic cycle of sarcoplasmic reticulum Ca2(+)-ATPase

The effect of a carboxylic ionophore (lasalocid) on the sarcoplasmic reticulum Ca2(+)-ATPase was investigated. The purified enzyme was preincubated with lasalocid in the presence of Ca2+ and the absence of K+ at pH 7.0 and 0 degrees C for 2 h. The Ca2(+)-dependent ATPase activity was strongly inhibited by this preincubation, whereas the activity of the contaminant Mg2(+)-ATPase was unaffected. The steady-state level of the phosphoenzyme (EP) intermediate remained constant over the wide range of lasalocid concentrations. The Ca2(+)-induced enzyme activation was unaffected. The kinetics of phosphorylation of the Ca2(+)-activated enzyme by ATP as well as the rate of conversion of ADP-sensitive EP to ADP-insensitive EP were also unaffected. Accumulation of ADP-insensitive EP was greatly enhanced, and almost all of the EP accumulating at steady state was ADP-insensitive. Hydrolysis of ADP-insensitive EP was strongly inhibited. A similar strong inhibition of the Ca2(+)-dependent ATPase activity by lasalocid was found with sarcoplasmic reticulum vesicles. To examine the effect of lasalocid on the conformational change in each reaction step, the Ca2(+)-ATPase of sarcoplasmic reticulum vesicles was labeled with a fluorescent probe (N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine) without a loss of catalytic activity and then preincubated with lasalocid as described above. The conformational changes involved in hydrolysis of ADP-insensitive EP and in the reversal of this hydrolysis were appreciably retarded by lasalocid. The conformational changes involved in other reaction steps were unaffected. These results demonstrate that hydrolysis of ADP-insensitive EP in the catalytic cycle of this enzyme is selectively inhibited by lasalocid.     

Distinct natures of beryllium fluoride-bound, aluminum fluoride-bound, and magnesium fluoride-bound stable analogues of an ADP-insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca2+-ATPase: changes in catalytic and transport sites during phosphoenzyme hydrolysis

The structural natures of stable analogues for the ADP-insensitive phosphoenzyme (E2P) of Ca(2+)-ATPase formed in sarcoplasmic reticulum vesicles, i.e. the enzymes with bound beryllium fluoride (BeF.E2), bound aluminum fluoride (AlF.E2), and bound magnesium fluoride (MgF.E2), were explored and compared with those of actual E2P formed from P(i) without Ca(2+). Changes in trinitrophenyl-AMP fluorescence revealed that the catalytic site is strongly hydrophobic in BeF.E2 as in E2P but hydrophilic in MgF.E2 and AlF.E2; yet, the three cytoplasmic domains are compactly organized in these states. Thapsigargin, which was shown in the crystal structure to fix the transmembrane helices and, thus, the postulated Ca(2+) release pathway to lumen in a closed state, largely reduced the tryptophan fluorescence in BeF.E2 as in E2P, but only very slightly (hence, the release pathway is likely closed without thapsigargin) in MgF.E2 and AlF.E2 as in dephosphorylated enzyme. Consistently, the completely suppressed Ca(2+)-ATPase activity in BeF-treated vesicles was rapidly restored in the presence of ionophore A23187 but not in its absence by incubation with Ca(2+) (over several millimolar concentrations) at pH 6, and, therefore, lumenal Ca(2+) is accessible to reactivate the enzyme. In contrast, no or only very slow restoration was observed with vesicles treated with MgF and AlF even with A23187. BeF.E2 thus has the features very similar to those characteristic of the E2P ground state, although AlF.E2 and MgF.E2 most likely mimic the transition or product state for the E2P hydrolysis, during which the hydrophobic nature around the phosphorylation site is lost and the Ca(2+) release pathway is closed. The change in hydrophobic nature is probably associated with the change in phosphate geometry from the covalently bound tetrahedral ground state (BeF(3)(-)) to trigonal bipyramidal transition state (AlF(3) or AlF(4)(-)) and further to tetrahedral product state (MgF(4)(2-)), and such change likely rearranges transmembrane helices to prevent access and leakage of lumenal Ca(2+).     

Purification of heterologous sarcoplasmic reticulum Ca2+-ATPase Serca1a allowing phosphoenzyme and Ca2+-affinity measurements.

We describe here a protocol to prepare milligrams of active and stable heterologous sarcoplasmic reticulum Ca(2+)-ATPase (Serca1a). Serca1a was tagged with 6 histidines at its C-terminal end and overexpressed using the baculovirus-Sf9 system. In a first trial, Serca1a accounted for 24% of membrane proteins, 95% of which were inactive. Glucose in the culture medium reduced the production of Serca1a to 3 to 5% of membrane proteins and all Serca1a was active. Seventy-five percent of active Serca1a was solubilized by C(12)E(8) in the presence of phosphatidylcholine under conditions avoiding denaturation. Purification by Ni(2+)-nitrilo-triacetic acid affinity chromatography was tried, but only 3% of active Serca1a remained bound to the column, as if the His-tag were not accessible. Yields of 43% were reached by purification on reactive red 120 columns when eluting with 2 M NaCl. The purity was about 25% and Serca1a was stable for at least 1 week at 0 degrees C. Typically, 500 ml of culture medium produced 3 mg of active Serca1a and 1 mg of purified active Serca1a allowing measurements of phosphoenzyme (2 nmol/mg) or Ca(2+) affinity (2 microM at pH 7).     

Critical hydrophobic interactions between phosphorylation and actuator domains of Ca2+-ATPase for hydrolysis of phosphorylated intermediate

Functional roles of seven hydrophobic residues on the interface between the actuator (A) and phosphorylation (P) domains of sarcoplasmic reticulum Ca2+-ATPase were explored by alanine and serine substitutions. The residues examined were Ile179/Leu180/Ile232 on the A domain, Val705/Val726 on the P domain, and Leu119/Tyr122 on the loop linking the A domain and M2 (the second transmembrane helix). These residues gather to form a hydrophobic cluster around Tyr122 in the crystal structures of Ca2+-ATPase in Ca2+-unbound E2 (unphosphorylated) and E2P (phosphorylated) states but are far apart in those of Ca2+-bound E1 (unphosphorylated) and E1P (phosphorylated) states. The substitution-effects were also compared with those of Ile235 on the A domain/M3 linker and those of T181GE of the A domain, since they are in the immediate vicinity of the Tyr122-cluster. All these substitutions almost completely inhibited ATPase activity without inhibiting Ca2+-activated E1P formation from ATP. Substitutions of Ile235 and T181GE blocked the E1P to E2P transition, whereas those in the Tyr122-cluster blocked the subsequent E2P hydrolysis. Substitutions of Ile235 and Glu183 also blocked EP hydrolysis. Results indicate that the Tyr122-cluster is formed during the E1P to E2P transition to configure the catalytic site and position Glu183 properly for hydrolyzing the acylphosphate. Ile235 on the A domain/M3 linker likely forms hydrophobic interactions with the A domain and thereby allowing the strain of this linker to be utilized for large motions of the A domain during these processes. The Tyr122-cluster, Ile235, and T181GE thus seem to have different roles and are critical in the successive events in processing phosphorylated intermediates to transport Ca2+.     

Calcium binding to the transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase: insights from molecular modeling

Sarcoplasmic reticulum Ca(2+)- ATPase pumps Ca(2+) ions from muscle cells to the sarcoplasmic reticulum. Here we use molecular dynamics and electrostatic modeling to investigate structural and dynamical features of key intermediates in the Ca(2+) binding process of the protein. Structural models of the protein (containing either two, one, or no calcium ions in the transmembrane domain) are constructed based on the X-ray structure by Toyoshima et al. (Nature 2000;405:647-655). The protein is embedded in a water/octane bilayer, which mimics the water/membrane environment. Our calculations provide information on the hydration of the two Ca(2+) ions, not emerging from the X-ray structure. Furthermore, they indicate that uptake of the metal ions causes large structural rearrangements of the metal binding sites. In addition, they suggest that the two ions reach their binding sites via two specific pathways. Finally, they allow identification of residues in the outer mouth of the protein that might interact with the Ca(2+) ions during the binding process.     

Multiple and distinct effects of mutations of Tyr122, Glu123, Arg324, and Arg334 involved in interactions between the top part of second and fourth transmembrane helices in sarcoplasmic reticulum Ca2+-ATPase: changes in cytoplasmic domain organization during isometric transition of phosphoenzyme intermediate and subsequent Ca2+ release

We explored, by mutational substitutions and kinetic analysis, possible roles of the four residues involved in the hydrogen-bonding or ionic interactions found in the Ca2+-bound structure of sarcoplasmic reticulum Ca2+-ATPase, Tyr(122)-Arg(324), and Glu(123)-Arg(334) at the top part of second transmembrane helix (M2) connected to the A domain and fourth transmembrane helix (M4) in the P domain. The observed substitution effects indicated that Glu(123), Arg(334), and Tyr(122) contributed to the rapid transition between the Ca2+-unbound and bound states of the unphosphorylated enzyme. Results further showed the more profound inhibitory effects of the substitutions in the M4/P domain (Arg(324) and Arg(334)) upon the isomeric transition of phosphorylated intermediate (EP) (loss of ADP sensitivity) and those in M2/A domain (Tyr(122) and Glu(123)) upon the subsequent processing and hydrolysis of EP. The observed distinct effects suggest that the interactions seen in the Ca2+-bound structure are not functionally important but indicate that Arg(334) with its positive charge and Tyr(122) with its aromatic ring are critically important for the above distinct steps. On the basis of the available structural information, the results strongly suggest that Arg(334) moves downward and forms new interactions with M2 (likely Asn(111)); it thus contributes to the inclination of the M4/P domain toward the M2/A domain, which is crucial for the appropriate gathering between the P domain and the largely rotated A domain to cause the loss of ADP sensitivity. On the other hand, Tyr(122) most likely functions in the subsequent Ca2+-releasing step to produce hydrophobic interactions at the A-P domain interface formed upon their gathering and thus to produce the Ca2+-released form of EP. During the Ca2+-transport cycle, the four residues seem to change interaction partners and thus contribute to the coordinated movements of the cytoplasmic and transmembrane domains.     

Critical interaction of actuator domain residues arginine 174, isoleucine 188, and lysine 205 with modulatory nucleotide in sarcoplasmic reticulum Ca2+-ATPase

ATP plays dual roles in the reaction cycle of the sarcoplasmic reticulum Ca2+-ATPase by acting as the phosphorylating substrate as well as in nonphosphorylating (modulatory) modes accelerating conformational transitions of the enzyme cycle. Here we have examined the involvement of actuator domain residues Arg174, Ile188, Lys204, and Lys205 by mutagenesis. Alanine mutations to these residues had little effect on the interaction of the Ca2E1 state with nucleotide or on the HnE 2 to Ca2E1 transition of the dephosphoenzyme. The phosphoenzyme processing steps, Ca2E1P to E2P and E2P dephosphorylation, and their stimulation by MgATP/ATP were markedly affected by mutations to Arg174, Ile188, and Lys205. Replacement of Ile188 with alanine abolished nucleotide modulation of dephosphorylation but not the modulation of the Ca2E1P to E2P transition. Mutation to Arg174 interfered with nucleotide modulation of either of the phosphoenzyme processing steps, indicating a significant overlap between the modulatory nucleotide-binding sites involved. Mutation to Lys205 enhanced the rates of the phosphoenzyme processing steps in the absence of nucleotide and disrupted the nucleotide modulation of the Ca2E1P to E2P transition. Remarkably, the mutants with alterations to Lys205 showed an anomalous inhibition by ATP of the dephosphorylation, and in the alanine mutant the affinity for the inhibition by ATP was indistinguishable from that for stimulation by ATP of the wild type. Hence, the actuator domain is an important player in the function of ATP as modulator of phosphoenzyme processing, with Arg174, Ile188, and Lys205 all being critically involved, although in different ways. The data support a variable site model for the modulatory effects with the nucleotide binding somewhat differently in each of the conformational states occurring during the transport cycle.     

The ADP- and Mg2+-reactive calcium complex of the phosphoenzyme in skeletal sarcoplasmic reticulum Ca2+-ATPase

The effects of ATP on Ca²⁺ binding in the absence of added Mg²⁺ to the purified sarcoplasmic reticulum Ca²⁺-ATPase were studied at pH 7.0 and 0°C. ATP increased the number of Ca²⁺-binding sites of the enzyme from 2 to 3 mol per mol of phosphorylatable enzyme. The association constant for the ATP-induced Ca²⁺ binding was 4·10⁵ M^?1, which was not significantly different from that obtained in the absence of ATP. AdoP[CH₂]PP has little effect on the Ca²⁺-binding process. The amount of phosphoenzyme formed was equivalent to the level of ATP-induced Ca²⁺ binding. ADP decreased the level of ATP-induced Ca²⁺ binding and phosphoenzyme by the same amount. These results suggest that ATP-induced Ca²⁺ binding exists in the form of an ADP-reactive phosphoenzyme·Ca complex. In addition, the Ca²⁺ bound to the enzyme in the presence of ATP was released on the addition of 1 mM MgCl₂; after the release of Ca²⁺, the phosphoenzyme decayed. These observations suggest that Mg²⁺, added after the ATP-induced Ca²⁺-binding process, may replace the Ca²⁺ on the phosphoenzyme and initiate phosphoenzyme decomposition.     

ADP-insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca(2+)-ATPase has a compact conformation resistant to proteinase K, V8 protease and trypsin

Sarcoplasmic reticulum Ca(2+)-ATPase was digested with proteinase K, V8 protease and trypsin in the absence of Ca(2+). Unphosphorylated enzyme was rapidly degraded. In contrast, ADP-insensitive phosphoenzyme formed with P(i) and phosphorylated state analogues produced by the binding of F(-) or orthovanadate, were almost completely resistant to the proteolysis except for tryptic cleavage at the T1 site (Arg(505)). The results indicate that the phosphoenzyme and its analogues have a very compact form in the cytoplasmic region, being consistent with large domain motions (gathering of three cytoplasmic domains). Results further show that the structure of the enzyme with bound decavanadate is very similar to ADP-insensitive phosphoenzyme. Thapsigargin did not affect the changes in digestion time course induced by the formation of the phosphorylated state analogues.     

Conformational changes of the Ca2+-ATPase as early events of Ca2+ release from sarcoplasmic reticulum

Rabbit skeletal sarcoplasmic reticulum vesicles were loaded with Ca2+ by ATP-dependent Ca2+ accumulation in the presence of low [Mg2+] (0.2-0.5 mM), and Ca2+ release was induced by addition of caffeine or ADP or by means of a Ca2+ jump. The levels of the phosphorylated intermediate (EP) and the tryptophan fluorescence of the Ca2+-ATPase were monitored during both the Ca2+ accumulation and the induced Ca2+ release using fast kinetic techniques. During Ca2+ uptake, both the EP level and the tryptophan fluorescence gradually decreased following a time course similar to that of the Ca2+ accumulation. Upon inducing Ca2+ release by addition of either caffeine or ADP, there was a transient increase of the EP level (from 0.3-0.5 to 1-2 nmol/mg protein) preceding the release of Ca2+. Similarly, a transient increase of the tryptophan fluorescence prior to Ca2+ release produced by the application of a Ca2+ jump was also found. These results indicate that the Ca2+-ATPase enzyme undergoes a rapid conformational change in response to triggering of Ca2+ release.     

Critical role of the first transmembrane domain of Cx26 in regulating oligomerization and function

To identify motifs involved in oligomerization of the gap junction protein Cx26, we studied individual transmembrane (TM) domains and the full-length protein. Using the TOXCAT assay for interactions of isolated TM α-helices, we found that TM1, a Cx26 pore domain, had a strong propensity to homodimerize. We identified amino acids Val-37-Ala-40 (VVAA) as the TM1 motif required for homodimerization. Two deafness-associated Cx26 mutations localized in this region, Cx26V37I and Cx26A40G, differentially affected dimerization. TM1-V37I dimerized only weakly, whereas TM1-A40G did not dimerize. When the full-length mutants were expressed in HeLa cells, both Cx26V37I and Cx26A40G formed oligomers less efficiently than wild-type Cx26. A Cx26 cysteine substitution mutant, Cx26V37C formed dithiothreitol-sensitive dimers. Substitution mutants of Val-37 formed intercellular channels with reduced function, while mutants of Ala-40 did not form functional gap junction channels. Unlike wild-type Cx26, neither Cx26V37I nor Cx26A40G formed functional hemichannels in low extracellular calcium. Thus the VVAA motif of Cx26 is critical for TM1 dimerization, hexamer formation, and channel function. The differential effects of VVAA mutants on hemichannels and gap junction channels imply that inter-TM interactions can differ in unapposed and docked hemichannels. Moreover, Cx26 oligomerization appears dependent on transient TM1 dimerization as an intermediate step.     

Characterization of the phosphoenzyme that is involved in the Ca2+ -Ca2+ exchange catalyzed by the Ca2+ -ATPase of sarcoplasmic reticulum vesicles

The rate of Ca2+ efflux was determined with 45Ca2+ -loaded sarcoplasmic reticulum vesicles (mainly with the light fraction of vesicles) at pH 6.5 and 0 degrees C. The efflux depended on external Ca2+, Mg2+, ATP and ADP, but it was not activated by AMP. The results indicate that the efflux is derived from Ca2+ -Ca2+ exchange mediated by the phosphoenzyme (EP) of membrane-bound Ca2+ -ATPase. EP was formed with Ca2+ -loaded vesicles (light fraction) under similar conditions without added ADP. The subsequent addition of EGTA and ADP induced triphasic EP dephosphorylation. Three species of EP (EP1, EP2, and EP3) were distinguished on the basis of this dephosphorylation kinetics, EP1, EP2, and EP3, corresponding to the first, second, and third phases of the dephosphorylation. Dephosphorylation of EP1 and EP2 resulted in stoichiometric ATP formation, while dephosphorylation of EP3 led to stoichiometric Pi liberation. The rate of Ca2+ efflux was compatible with that of EP2 dephosphorylation, whereas it was much lower than the rate of EP1 dephosphorylation and much higher than the rate of EP3 dephosphorylation. The intravesicular Ca2+ concentration dependence of the rate of EP2 dephosphorylation agreed with that of the rate of Ca2+ efflux. The results suggest that isomerization between EP1 and EP2 is the rate-limiting process in the Ca2+ -Ca2+ exchange and that EP3 is not involved in this exchange.     

Characterization of the phosphoenzyme that is involved in the Ca2+-Ca2+ exchange catalyzed by the Ca2+-ATPase of sarcoplasmic reticulum vesicles

The rate of Ca²⁺ efflux was determined with ⁴⁵Ca²⁺-loaded sarcoplasmic reticulum vesicles (mainly with the light fraction of vesicles) at pH 6.5 and 0°C. The efflux depended on external Ca²⁺, Mg²⁺, ATP and ADP, but it was not activated by AMP. The results indicate that the efflux is derived from Ca²⁺-Ca²⁺ exchange mediated by the phosphoenzyme (EP) of membrane-bound Ca²⁺-ATPase. EP was formed with Ca²⁺-loaded vesicles (light fraction) under similar conditions without added ADP. The subsequent addition of EGTA and ADP induced triphasic EP dephosphorylation. Three species of EP (EP₁, EP₂, and EP₃) were distinguished on the basis of this dephosphorylation kinetics, EP₁, EP₂ and EP₃, corresponding to the first, second, and third phases of the dephosphorylation. Dephosphorylation of EP₁ and EP₂ resulted in stoichiometric ATP formation, while dephosphorylation of EP₃ led to stoichiometric P_i liberation. The rate of Ca²⁺ efflux was compatible with that of EP₂ dephosphorylation, whereas it was much lower than the rate of EP₁ dephosphorylation and much higher than the rate of EP₃ dephosphorylation. The intravesicular Ca²⁺ concentration dependence of the rate of EP₂ dephosphorylation agreed with that of the rate of Ca²⁺ efflux. The results suggest that isomerization between EP₁ and EP₂ is the rate-limiting process in the Ca²⁺-Ca²⁺ exchange and that EP₃ is not involved in this exchange.