Pumps SERCA

The only known mechanism for Ca2+ accumulation by the endoplasmic reticulum is by means of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pumps 381... 

The plasma membrane Ca2+-ATPase pump effects outward transport of Ca2+ against a large electrochemical gradient for Ca2+. The mechanism of the pump involves its phosphorylation by ATP and the formation of a high-energy intermediate. This basic mechanism is similar for both the plasma membrane and ER pumps however, the structures of these distinct gene products are substantially different. As discussed below, the ER pump, sometimes called a sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pump, is inhibited potently by certain natural and synthetic toxins that do not affect the plasma membrane pump. The plasma membrane pump, but not the SERCA pump, is controlled in part by Ca2+ calmodulin, allowing for rapid activation when cytoplasmic Ca2+ rises. 

Sanders The same thing happened with thapsigargin. It s as if CPA were doing something besides blocking SERCA pumps. It doesn t always match with thapsigargin. 

The Ca2+ taken up in the TG-insensitive compartment could be released by the Ca2+ ionophore A23187. Only a partial release was observed with L1SP3. The concentration of InsP3 required for half-maximal release was about two times higher than that required for half-maximal activation of Ca2+ release from the compartment filled by the SERCA pump. A very similar pattern was seen for the InsP3 effect on the additional Ca2+ compartment created in COS-1 cells by overexpression of the worm Pmrl. Although still indirect, these data strongly... 

Blaustein Some areas empty and others fill when we put on CPA. This means that there are some SERCA Ca2+ pumps that are insensitive to CPA. A number of studies have demonstrated that there are some SERCA pumps that are insensitive. If you dump Ca2+ out of some regions and other Ca2+ pumps are still working, you are going to fill those regions. 

Paul It will inhibit the SERCA pump. Again, we don t know at what level, nor do we know what the binding capacity is. Even to model it at this stage might be difficult. 

The main mechanisms that are important are (1) SERCA pumps (2) phospholamban (3) Ca2+ binding proteins (4) inositohl,4,5-trisphosphate (L1SP3) receptors and mechanisms involved in InsP3 production (5) ryanodine receptors and cADP ribose production and (6) the cytoskeleton. 

Ca2+ is pumped into the SR stores by specific ATPases, the SERCA pumps. Three SERCA genes have been identified and several alternatively spliced gene products. In muscles SERCA1 is predominantly expressed in fast-twitch striated muscles and SERCA 2 isoforms are dominant in slow twitch, heart and smooth muscles, with SERCA2a in the former two, and SERCA2b predominant in smooth muscle and non-muscle cells. SERCA3 has a widespread distribution. 

Phospholamban is a protein that inhibits the SERCA pumps by decreasing their affinity for Ca2+. It can be phosphorylated by protein kinase A, for instance in response to /1-adrenoceptor activation, resulting in inhibition of its effects and enhanced SERCA activity. The effects of phospholamban on contraction depend on the relative importance of Ca2+ uptake or release in the smooth muscle in... 

Several of the proteins that mediate Ca2+ flow in and out of SR have been identified. Oxalate-facilitated Ca2+ uptake into the SR and in vitro biochemical studies of purified SR identified it as an ATP-driven Ca2+ pump (SERCA pump reviewed in Himpens et al 1995) that is inhibited by thapsigargin and cyclopiazonic acid and regulated, at least in some smooth muscles, by phosphorylation of phospholamban by cyclic nucleotide-activated protein kinase(s) (Karczewski et al 1998). 

The ER takes up Ca2+ using a sarco(endo)plasmic reticulum ATPase (SERCA) pump (Figure 11.5), which has the same topology with 10 transmembrane domains and the same mechanism of action, with an aspartyl phosphate intermediate, as the PMCA pump described above. 

The SERCA pump of many types of muscle is regulated by a protein called phospholam-bin, which binds to SERCA in both its cytosolic and its transmembrane regions, maintaining the pump in an inactivated state when in its non-phosphorylated form, but detaches from... 

Figure 11.5 Three-dimensional structure of the SERCA pump in the El (Ca2+-bound) and E2 (Ca2+-free) state. (From Carafoli, 2005. Reproduced with permission from Blackwell Publishing Ltd.)...

The Golgi apparatus also appears to be an important regulator of intracellular Ca2+ homeostasis. It has a SERCA pump, as well as an unusual Ca2+-ATPase that can transport Ca2+ and Mn2+ with high affinity for Ca2+ uptake. Ca2+ release is mediated by a channel that is modulated by InsP3, similar to that in the ER. However, it does not seem to have ryanodine-type channels. 

The cytosolic concentration of free Ca2+ is generally at or below 100 mi, far lower than that in the surrounding medium, whether pond water or blood plasma. The ubiquitous occurrence of inorganic phosphates (Pj and I l ,) at millimolar concentrations in the cytosol necessitates a low cytosolic Ca2+ concentration, because inorganic phosphate combines with calcium to form relatively insoluble calcium phosphates. Calcium ions are pumped out of the cytosol by a P-type ATPase, the plasma membrane Ca2+ pump. Another P-type Ca2+ pump in the endoplasmic reticulum moves Ca2+ into the ER lumen, a compartment separate from the cytosol. In myocytes, Ca2+ is normally sequestered in a specialized form of endoplasmic reticulum called the sarcoplasmic reticulum. The sarcoplasmic and endoplasmic reticulum calcium (SERCA) pumps are closely related in structure and mechanism, and both are inhibited by the tumor-promoting agent thapsigargin, which does not affect the plasma membrane Ca2+ pump. 

The amino acid sequences of the SERCA pumps and the Na+K+ ATPase share 30% identity and 65% sequence similarity, and their topology relative to the membrane is also the same. Thus it seems likely that the Na+K+ ATPase structure is similar to that of the SERCA pumps and that all P-type ATPase transporters share the same basic structure. 

P-type ATPases 398 SERCA pump 400 F-type ATPases 401 ATP synthase 401 V-type ATPases 401 ABC transporters 402 multidrug transporter 402... 

The SERCA pumps belong to the P-type ATPase family, which actively transport cations across membranes at the expense of ATP hydrolysis. They show a high degree of conservation among species and their structure has recently been... 

In humans, SERCA pumps are encoded by three genes, ATP2A1-3, each encoding several protein isoforms (SERCAla,b, SERCA2a-c, SERCA3a-f, respectively) as a result of developmental or tissue-specific alternative splicing (Table 1). 

Figure 3. Schematic representation of the reaction cycle of SERCA pumps. The SERCA pumps exist in two conformational state El binds Ca2+ with high affinity at the cytoplasmic site of the SER membrane, while E2 has low affinity for Ca2+ and thus releases it on the opposite site of the membrane. ATP phosphorylates a highly conserved aspartic acid residue allowing for the translocation of Ca2+ in the SER lumen...

SERCA pumps in cultured COS cells. Their functional properties were studied using isolated microsomes. The initial in vitro studies reported that SERCA1 and SERCA2a isoforms shared similar Ca2+ affinity and velocity of Ca2+ uptake (Vmax). Subsequently, a higher kinetic turnover was demonstrated for the SERCA1 compared with the SERCA2a isoform (5.0- versus 2.6-fold increase in calcium uptake rate) (Sumbilla et al 1999). 

SERCA pumps are essential actors of the Ca2+ signaling pathway in excitable and non excitable cells. Following cell stimulation, they restore low Ca2+ cytosolic concentrations and contribute to generate intracellular Ca2+signals leading to specific cell responses. They reconstitute dynamic Ca2+ stores in the SR/ER required for post-translational modifications, for quality control in the SR/ER and for subsequent Ca2+ release. 

SERCA pumps sequester Ca2+ in the ER lumen By maintaining appropriate Ca2+ concentrations in the ER lumen, SERCA pumps also play an essential role in protein synthesis, folding and transport of membrane and secreted proteins. This involves in particular chaperone-dependent processing and post-translational modifications which require a unique calcium rich environment. Chaperone molecules such as calreticulin and calnexin are involved in the quality control pathway in the ER (Berridge, 2002 Ellgaard and Helenius, 2003 Michalak et al., 2002). 

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