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 f unctional 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) in hibition 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 observat ions and molecular models revealed short and long range functions of s everal amino acids within the transmembrane region, Short range functi ons include: (a) direct involvement of five amino acids in Ca2+ bindin g within a channel formed by clustered transmembrane helices M4, M5, M 6, and M8; (b) roles of several amino acids in structural stabilizatio n of the helical cluster for optimal channel function; and (c) a speci fic role of Lys(297) in sealing the distal end of the channel, suggest ing that the M4 helix rotates to allow vectorial flux of Ca2+ upon enz yme phosphorylation, Long range functions are related to the influence of several transmembrane amino acids on phosphorylation reactions wit h ATP or P-i, transmitted to the extramembranous region of the ATPase in the presence or in the absence of Ca2+.