View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library Basic Res Cardio192: Suppl. 1, 59 - 61 SteinkopffVerlag 1997 E. Carafoli Plasma membrane calcium pump: structure, function and relationships Abstract The plasma membrane spicing. Most of the pump mass prot- (N-terminal) protruding unit. The Ca-pump (134 kDa) is stimulated by rudes into the cytoplasm with three isoforms of the pump show variations calmodulin and by other treatments main units. The calmodulin binding in the regulatory domains, e.g., alter- (exposure to acidic phospholipids, domain is located in the C-terminal native mRNA splicing can eliminate treatments with proteases, phos- protruding unit. The domain is a the domain phosphorylated by pro- phorylation by protein kinases A or positively charged segment of about tein kinase A, or alter the sensitivity C, self-association to form oligom- 25 residues. The calcium-activated of the pump to calmodulin. This ers). It is the product of four genes protease calpain activates the pump occurs by inserting sequences rich in (in humans), but additional isoforms by removing its calmodulin binding His between calmodulin binding originate through alternative mRNA domain and the portion C-terminal subdomains A and B. The inserted to it. The-resulting 124 KDa fragment domain(s) confer pH sensitivity to has been used to test the suggestion the binding of calmodulin. Calcium of an autoinhibitory function of the binding sites have been found in Ernesto Carafoli (5:~) calmodulin binding domain. The acidic regions preceding and follow- Laboratory of Biochemistry III latter interacts with two domains of ing the calmodulin binding domain. Swiss Federal Institute of Technology (ETH), the pump, one located close to the Universit~tsstr. 16 active site in the mid-cytoplasmic Key words Calcium pump - 8092 Ztirich, Switzerland protruding unit, the other in the first calmodulin - calpain The plasma membrane Ca-pump (PMCA) is the largest of exposure to acidic phospholipids (including the phos- all P-type ion pumps, i.e., pumps which form a phos- phorylated derivatives of phosphatidyl-inositol), a con- phorylated intermediate in the catalytic cycle (an aspar- trolled treatment with proteases, e.g., trypsin or calpain, tyl-phosphate) and are inhibited by vanadate. The PMCA phosphorylation reactions by protein kinases A or C and pump interacts with Ca with high affinity, but has a low self-association to form dimers or, more likely, oligomers. total Ca transporting capacity. This property makes it Cloning work (4, 8) in the late 1980s has shown that the inadequate to rapidly eject large amounts of Ca, as pump is a single polypeptide chain of molecular mass required, for exampie, by the functional cycle of heart about 134 KDa. It is the product of a multigene family: cells. Thus, in these cells, the pump is quantitatively over- four genes have been recognized in humans, and they shadowed by a larger system, the NaCa exchanger. First have been assigned to chromosomes 1, 3, 12 and x. Numer- discovered in erythrocytes, the PMCA pump has been ous additional isoforms then originate through alternative subsequently found in many other cells, and is now mRNA spicing. The general architecture of the pump assumed to be an obligatory component of eucaryotic (Fig. 1) follows the general pattern of the P-type pump plasma membranes. It is stimulated by calmodulin and, in family, i.e., most of the pump mass protrudes into the its absence, by a number of alternative treatments like the cytoplasmic medium, very short loops connecting the 60 Basic Research in Cardiology,Vot. 92, Suppl. 1 (1997) 9Steinkopff Verlag 1997 putative transmembrane domain on the external side. Ten the last protruding unit of the pump. The domain is a posi- putative transmembrane domains have been identified, tively charged segment of about 25 residues, which has a four concentrated in the N-terminal portion of the pump strong s-helix propensity in its N- and C-terminal portions and six in the C-terminal portion. The transmembrane (sub-domains A and B). Work with trypsin has shown that organization of the pump has been, to a large part, the pump is sequentially degraded to fragments of elec- directly validated by work with monoclonal antibodies trophoretic molecular masses 90, 85, 81, 76 KDa. The 90 and with other chemical labelling techniques. Three main KDa fragment has low basal activity and is stimulated by units protrude into the cytosol, the first between trans- both calmodulin and acidic phospholipids. During the for- membrane domains 2 and 3, the second between trans- mation of the 85 and 81 KDa fragments, the pump gradu- membrane domains 4 and 5, and the third from transmem- ally loses its sensitivity to calmodulin, but retains that to brane domain 10. The first protruding unit has been prop- acidic phospholipids, whereas the formation of the 76 osed, based on the analogy with other P-type pumps, to KDa fragment is accompanied by the loss of phospholipid permit the coupling of ATP hydrolysis to the transport of sensitivity as well. The removal of the calmodulin binding Ca and is thus sometimes called the transducing unit. The domain and of the pump portion C-terminal to it during protruding portions of the pump (Fig. 1) consist of anti- the formation of these fragments leads to the stimulation parallel /~-sheets, a-helices and parallel /3-sheets. The of the basal activity of the pump, suggesting that the domain protruding between transmembrane segments 4 domain acts as an autoinhibitor of the pump. In the forma- and 5 contains the active site(s) of the pump (i.e., the site tion of the 90 to 76 KDa fragments, trypsin also cuts the of aspartyl-phosphate formation and the binding site for pump between putative transmembrane domains 2 and 3, ATP), and is assumed to be connected to the membrane but it is doubtful whether the portion of the pump N-ter- by a flexible "hinge" in its C-terminal portion which per- minal to the cut is actually removed. The Ca-dependent mits the catalytic aspartic acid to approach the bound ATP protease calpain also activates the pump, and does so by during the reaction cycle. removing in two steps its calmodulin binding domain and The calmodulin binding domain of the pump has been the portion C-terminal to it. The resulting 124 KDa frag- identified with the help of bifunctional crosslinkers ment has been used to test the suggestion of the auto- coupled to calmodulin (James et al., 1988), and located in inhibitory function of the calmodulin binding domain. The latter, suitably derivatized with a photoactivatable crosslinker (2) has been found to label two domains of the pump, one located between the sites of aspartyl-phos- phate formation and of ATP-binding, the other in the first protruding unit of the pump. The finding that the "recep- tor" sites for the calmodulin binding domain are located in close proximity to the active site(s) of the pump conve- niently rationalizes the "inhibitory', action of the domain. The calmodulin binding domain can be phosphorylated by protein kinase C, leading to some activation of the basal activity of the pump (9). The calmodulin-binding domain has been prepared synthetically with a phosphate on the threonine that is the substrate for protein kinase C (6): the phosphorylated domain is unable to inhibit the fully active 124 kDa calpain product. This establishes a striking parallel with the Ca-pump of sarcoplasmic reticulum which is kept inhibited by the association of the accessory protein, phospholamban, and which is reacti- vated by the removal of phospholamban from its binding site by kinase-directed phosphorylations. Fig. 1 Membranearchitecture and domain definitionof the PMCA pump. Cylindersindicate predicted a-helices, arrows/3-sheets. The On the N-terminal side, trypsin cleaves the pump two calmodulinbinding sub-domainsare indicatedby A and B. Calp between transmembrane domains two and three. The N- I and calp 2 are the two sequential sites of calpain cleavage. PKA (S) termini of the fragments of 90, 85, 81 KDa differ from that and PKC (T) are sites of serine and threonine kinase phosphoryla- of the 76 KDa fragment by a heavily charged stretch of tion: PKA only phosphorylatesone of the isoformsof the pump. TC is the notationfor the C-terminal cleavage by trypsin,TN that of the about 40 residues. Work with the synthetic C-terminal por- N-terminal cleavage. PL is the main domain responsive to acidic tion of the stretch, which is very rich in basic amino acids phospholipids. (labelled PL in Fig. 1), has shown (1) it to be one of the E. Carafoli 61 Plasma membrane calcium pump: structure, function and relationships sites of the pump responsible for the response to acidic suggestions on the Ca pump of sarcoplasmic reticulum, phospholipids (the other site appears to be the calmodulin this Ca binding site could be located within some of the binding domain itself). transmembrane domains. Mutagenesis work on selected None of the isoforms of the pump show variations in residues in some of the transmembrane domains are in the domains which are preserved throughout the family of line with the prediction. Other Ca binding sites, possibly P-type ion motive ATPases, e.g., the domains surround- regulatory, have recently been found immediately C- and ing the active site(s). Most of the diversity concerns the N-terminal to the calmodulin binding domain (6). regulatory domains and leads to differences in regulatory Despite the similarity of the plasma membrane and the properties, e.g., alternative mRNA splicing can eliminate endoplasmic reticulum Ca pumps, the two enzymes are the domain phosphorylated by protein kinase A (PKA(S) targeted to two different membranes.