SITES OF REGULATORY INTERACTION BETWEEN CALCIUM ATPASES AND PHOSPHOLAMBAN

In an effort to define the amino acids that are involved in functional interactions between phospholamban (PLN) and the Ca2+ ATPase of cardi ac sarcoplasmic reticulum (SERCA2), we have coexpressed wild type and mutant forms of phospholamban with wild type and mutant forms of SERCA 2, isolated microsomal fractions and measured Ca2+ dependence of Ca2transport. We have found that both charged and hydrophobic residues in the cytoplasmic domains of both PLN and SERCA2 make up the cytoplasmi c interaction site. In SERCA2, this site is the linear sequence Lys-As p-Asp-Lys-ProVal(402): In PLN, the site is more diffuse and complex. F unction was retained if the net charge over the first 20 amino acids w as +1 or +2, but function was lost if the net charge was -3, -2, 0 or +3. Function was also lost if the long alkyl side chains of Val(4), Le u(7) or Ile(12) were replaced with the methyl group of Ala. We have al so obtained evidence that a site of functional interaction is present in the transmembrane domains of PLN and SERCA2.