Short and long range functions of amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase. A mutational study.
Mutational analysis of several amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase was performed by expressing wild type ATPase and 32 site-directed mutants in COS-1 cells followed by functional characterization of the microsomal fraction. Four different phenotype characteristics were observed in the mutants: (a) functions similar to those sustained by the wild type ATPase; (b) Ca2+ transport inhibited to a greater extent than ATPase hydrolytic activity; (c) inhibition of transport and hydrolytic activity in the presence of high levels of phosphorylated enzyme intermediate; and (d) total inhibition of ATP utilization by the enzyme while retaining the ability to form phosphoenzyme by utilization of P(i). Analysis of experimental observations and molecular models revealed short and long range functions of several amino acids within the transmembrane region. Short range functions include: (a) direct involvement of five amino acids in Ca2+ binding within a channel formed by clustered transmembrane helices M4, M5, M6, and M8; (b) roles of several amino acids in structural stabilization of the helical cluster for optimal channel function; and (c) a specific role of Lys297 in sealing the distal end of the channel, suggesting that the M4 helix rotates to allow vectorial flux of Ca2+ upon enzyme phosphorylation. Long range functions are related to the influence of several transmembrane amino acids on phosphorylation reactions with ATP or P(i), transmitted to the extramembranous region of the ATPase in the presence or in the absence of Ca2+.
Chen
L
L
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore 21201, USA.
Sumbilla
C
C
Lewis
D
D
Zhong
L
L
Strock
C
C
Kirtley
M E
ME
Inesi
G
G
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D002645:Chickens; D018076:DNA, Complementary; D007425:Intracellular Membranes / Q000201:enzymology; D017136:Ion Transport; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000596:Amino Acids; D018076:DNA, Complementary; D010710:Phosphates; D000252:Calcium-Transporting ATPases; D002118:Calcium