Chapter 4 Sodium-calcium exchangers and calcium pumps

Ca²⁺ acts as a universal second messenger in intracellular signaling in eukaryotes. The maintenance and regulation of the free intracellular Ca²⁺ concentration requires the presence of specialized Ca²⁺ transporters in the plasma membrane and in intracellular organellar membranes. Na⁺/Ca²⁺ exchangers and ATP-driven Ca²⁺ pumps are the two major systems responsible for uphill Ca²⁺ transport against its large concentration gradient. Na⁺/Ca²⁺ exchangers are high capacity transporters involved in the handling of large and dynamic Ca²⁺ fluxes across the plasma membrane. Cardiac-type exchangers are ubiquitous but are most abundant in excitable cells such as in heart and nervous tissue. They use the energy stored in the electrochemical Na⁺ gradient to exchange one Ca²⁺ for three Na⁺ per reaction cycle. In the forward mode they expel Ca²⁺ from the cell in exchange for Na⁺. However, depending on the prevailing Na⁺ and Ca²⁺ ion distribution and on the membrane potential they can also operate in the reverse mode. The photoreceptor exchanger is highly specific for the outer membrane segment of vertebrate retinal rods and normally operates with a 4Na⁺/1Ca²⁺, 1K⁺ stoichiometry. The cardiac-and photoreceptor-type exchangers are single polypeptides with native molecular masses of about 120 and 220 kDa, and cDNA-derived calculated masses of about 105 and 130 kDa, respectively. The differences are mainly due to glycosylation on their N-terminal extracellular segments. Both exchanger types contain a leader peptide which is cleaved during maturation, and they show extensive structural similarities in their predicted transmembrane topology: two clusters of five and six transmembrane segments close to the N-and C-termini, respectively, are separated by a large cytosolic hydrophilic domain. Ca²⁺ pumps are divided into two distinct families of plasma membrane and sarco/endoplasmic reticulum membrane Ca²⁺ ATPases (PMCAs and SERCAs). In tissues with a low Na⁺/Ca²⁺ exchanger activity and under conditions unfavorable for Na⁺/Ca²⁺ exchange, PMCAs are the sole mechanism of specific Ca²⁺ export. They play an essential role in the long-term maintenance and the resetting of resting free Ca²⁺ levels. SERCAs are responsible for the rapid Ca²⁺ reuptake into the endoplasmic/sarcoplasmic reticulum and its specialized compartments after Ca²⁺ signaling. Ca²⁺ pumps belong to the class of P-type pumps which are characterized by the formation of a phosphorylated intermediate in which the energy derived from ATP is transiently stored as conformational constraint. Ca²⁺ pumps shuttle between two major conformational states E₁ and E₂. In the E₁ state they bind Ca²⁺ on the cytosolic (cis) side with very high affinity (K_Ca≈0.5 μM). Upon Ca²⁺ binding their ATPase activity is stimulated, leading to the formation of the phosphorylated intermediate and followed by occlusion of the transported Ca²⁺, transition to the low energy, low Ca²⁺ affinity E₂ state and release of Ca²⁺ on the trans side. Two Ca²⁺ ions are transported per ATP split in SERCAs but only one in the PMCAs. SERCAs and PMCAs are single polypeptides with molecular masses of about 100 and 130 kDa. Despite limited primary sequence identity their predicted transmembrane topologies are very similar and consits of two clusters of four and six transmembrane segments separated by a large cytosolic domain containing the catalytic and ATP binding sites. Polar amino acid side chains within at least four transmembrane segments participate in forming the high affinity Ca²⁺ binding/transport sites in SERCAs, and probably in PMCAs, too. Long-range conformational interactions must occur in the pumps to enable changes in the catalytic site region to affect Ca²⁺ binding and translocation in the transmembrane region. PMCAs, but not SERCAs, are directly stimulated by Ca²⁺-calmodulin; indeed, most of the additional mass of PMCAs with respect to SERCAs is due to a C-terminal regulatory region containing the autoinhibitory, calmodulin-binding domain. Multiple isoforms of SERCAs and PMCAs are generated from multigene families and from alternatively spliced mRNAs. The tissue and developmental stage-specific expression of several SERCA and PMCA isoforms suggests that specific functional and/or regulatory properties of these pumps are adapted to the physiological needs of a given cell type.