Transmembrane helix M6 of the sarco(endo)plasmic reticulum Ca 2؉ ATPase (SERCA) has been shown to form a site of interaction with phospholamban (PLN). Site-directed mutagenesis was carried out in the cytoplasmic loop (L67) between M6 and M7 in SERCA1a to detect other SERCA-PLN binding sites. Mutants N810A, D813A, and R822A had diminished ability to interact functionally with PLN, but only D813A and R822A had reduced physical interaction with PLN. PLN mutants R25A, Q26A, N27A, L28A, Q29A, and N30A had enhanced physical interaction with wild-type (wt) SERCA1a, but physical interaction of these PLN mutants with SERCA1a mutants D813A and R822A was reduced about 2.5 fold (range 1.44 -2.82). Exceptions were the interactions of PLN N27A and N30A with SERCA1a D813A, which were reduced by 7.3-and 5.8-fold, respectively. A superinhibitory PLN deletion mutant, PLN⌬21-29, had strong physical interactions with SERCA1a and with SERCA1a mutant D813A. Physical interactions with SERCA1a and mutant D813A were sharply diminished, however, for the PLN deletion mutant, PLN⌬21-30, lacking PLN N30. Physical interactions between SERCA1a and a PLN-cytochrome b 5 chimera containing PLN residues 1-29 were much stronger than those between a PLN-cytochrome b5 chimera containing PLN residues 1-21 and lacking N27. These results suggest that a SERCA1-PLN interaction site occurs between L67 of SERCA1a and domain IB of PLN, which involves SERCA1a D813 and PLN N27 and N30. . The phosphorylation and nucleotide binding domains resemble the major and subdomains of the haloacid dehalogenase (4). Unexpected features that are of obvious functional significance are the association of two helices at the N terminus of the molecule with a cytosolic ␤-strand domain between helices M2 and M3 to form an actuator domain and a cytosolic loop (L67) between M6 and M7 (F809-G831), which winds around and links together several elements of the stalk sector. These two features set up linkages that might involve virtually all of the transmembrane helices in the conformational movements that drive Ca 2ϩ transport.The role of amino acid residues in the L67 loop in Ca 2ϩ transport has been investigated through site-directed mutagenesis (5, 6). Mutations K819A and R822A inhibit Ca Phospholamban (PLN) is a 52-aa integral membrane protein, highly expressed in cardiac and slow-twitch skeletal muscle fibers, that interacts with and, at low Ca 2ϩ concentrations, reversibly inhibits the activity of SERCA2a by lowering its apparent affinity for Ca 2ϩ (7). Studies of mice in which PLN is ablated or replaced by highly inhibitory forms of PLN demonstrate that PLN is a modulator of the activity of the Ca 2ϩ pump and a major regulator of the dynamics of cardiac contractility (8-11). It has been proposed that PLN contains three domains. Domain IA, amino acids 1-20, contains sites of regulatory phosphorylation by protein kinase A at S16 and by Ca 2ϩ -calmodulin kinase at T17; domain IB, amino acids 21-30, contains a high proportion of amidated residues; domain II, amino acids 31-52, fo...