Roles of Tyr122-hydrophobic Cluster and K+ Binding in Ca2+-releasing Process of ADP-insensitive Phosphoenzyme of Sarcoplasmic Reticulum Ca2+-ATPase

Kazuo Yamasaki,Guoli Wang,Takashi Daiho,Stefania Danko,Hiroshi Suzuki

doi:10.1074/jbc.m804596200

Kazuo Yamasaki, Guoli Wang + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m804596200

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Abstract

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.

Highlights

We found that mutations in a specific hydrophobic interaction network, “Tyr122-hydrophobic cluster” (Y122-HC), at the A-P domain interface disrupt markedly the processing of ADP-insensitive EP formed from ATP with Ca2ϩ and the hydrolysis of E2P formed from Pi without Ca2ϩ, causing nearly complete inhibition of the Ca2ϩ-ATPase activity [22, 23]
In the mutant Y122A, the fraction of the ADP-insensitive EP was very high at the low Ca2ϩ concentrations at all pHs (Fig. 2). This agrees with the property of this mutant [22, 23] that the hydrolysis of E2P is nearly completely inhibited causing its accumulation
The results suggested that the lumenal Ca2ϩ affinity of transport sites of E2P in the mutant may be significantly higher than that in the wild type

Summary

EXPERIMENTAL PROCEDURES

Mutagenesis and Expression—Mutations were created by the QuikChangeTM site-directed mutagenesis kit (Stratagene) and plasmid pGEM7-Zf(ϩ) or pGEM3-Zf(ϩ) (Promega, Madison, WI) containing ApaI-KpnI or KpnI-SalI fragments of rabbit SERCA1a cDNA as a template. The ApaI-KpnI or KpnI-SalI fragments were excised from the products and used to replace the corresponding region in the full-length SERCA1a cDNA in the pMT2 expression vector [25]. Microsomes were prepared from the cells as described previously [26]. The “control microsomes” were prepared from COS-1 cells transfected with the pMT2 vector containing no SERCA1a cDNA. ATPase Activity—The rate of ATP hydrolysis was determined at 25 °C in a mixture containing 20 ␮g/ml microsomal. Formation and Hydrolysis of EP—Phosphorylation of SERCA1a in microsomes with [␥-32P]ATP or 32Pi and dephosphorylation of 32P-labeled SERCA1a were performed under conditions described in the figure legends. The radioactivity associated with the separated Ca2ϩ-ATPase was quantitated by digital autoradiography as described previously [29]. The amount of EP formed with the expressed SERCA1a was obtained by subtracting the background radioactivity with the control microsomes. Three-dimensional models of the enzyme were reproduced by using the program VMD [31]

RESULTS

WT ϩ

DISCUSSION