Single amino acid mutations in transmembrane domain 5 confer to the plasmamembrane Ca2+ pump properties typical of the Ca2+ pump of endo(sarco)plasmic reticulum

Conserved residues in some of the transmembrane domains are proposed to med iate ion translocation by P-type pumps. The plasma membrane Ca2+ pump (PMCA ) lacks 2 of these residues in transmembrane domains (TM) 5 and 8, In parti cular, a glutamic acid (Glu-771) residue in TM5, which is proposed to be in volved in the binding and transport of Ca2+ by the sarcoplasmic reticulum C a2+ pump (SERCA), is replaced by an alanine (Ala-854) in the PMCA pump. Ala -854 has been mutated to Glu, Asp, or Gin; Glu-975 in TM8, which is an Ala in the SERCA pump, has been mutated to Gin, Asp, or Ala, The mutants have b een expressed in three cell systems, with or without the help of viruses. W hen expressed in large amounts in Sf9 cells, the mutated pumps were isolate d and analyzed in the purified state. Two of the three TM8 mutants were cor rectly delivered to the plasma membrane and were active. AU the TM5 mutants were retained in the endoplasmic reticulum; two of them (A854Q and A854E) retained activity. Their properties (La3+ sensitivity and decay of the phos phorylated intermediate, higher cooperativity of Ca2+ binding with a Hill's coefficient approaching 2) differed from those of the expressed wild type PMCA pump, and resembled those of the SERCA pump.