Calcium Pumping: P–type ATPase

 

Structure

 

The Calcium ATPase is composed of 10 transmembrane alpha helices 3 of which line the central channel through the lipid bilayer. Connected to the helices are 3 specific domains that reach into the cytoplasm each of which have a specific function.

  1. -N Domain (nucleotide binding domain)

  1. -P Domain (phosphorylation domain)

  1. -A Domain (activator domain)

  

While calcium ATPase is unphosphorolated two of the helices are disrupted forming a cavity accessible from the cytosol which binds 2 Ca 2+ ions. ATP binds to a site which phosphorolates the adjacent P domain. The binding and hydrolysis of ATP causes conformational changes which bring the N and P domains into close proximity. This then causes a 90 degree rotation of the A domain which causes the transmembrane helices 4 and 6 to rearrange. This then releases the Ca into the lumen of the sarcoplasmic reticulum.

  

 

 

  

Function & Role

 

A whole range of cellular processes are regulated by the free cytosolic calcium concentration, ranging from transcription control and cell survival to neurotransmitter release and muscle function. In order for a cell to use calcium as a signaling molecule, the cell must create calcium gradients across membranes. In order to obtain such concentration differences, calcium ions need to be actively pumped across membranes against a concentration gradient. For this reason cells use calcium pumps to direct the flow of calcium ions through the plasma membrane or organelle membranes and the resulting gradients are used in a variety of signaling systems mediated by gated channels.

 

Calcium pumps are ATPases that transport ions across membranes using energy obtained from the hydrolysis of ATP. Calcium ATPases are members of the P-type family of ion pumps, which are responsible for the ATP dependent active transport of ions across a wide variety of cellular membranes. The designation of P-type comes from the mechanism, which involves the phosphorylation of an aspartate residue (Asp351) in the active site using the terminal phosphate in ATP, resulting in conformational changes in both the ATP-binding cytoplasmic domains and the calcium binding transmembrane domain that shuffles the ions across the membrane. The subsequent release of the calcium ions signals the hydrolysis of the aspartyl-phosphate group, returning the pump to its original conformation.

 

The basic function of the plasma membrane calcium pump is to maintain the 10,000-fold calcium gradient across the plasma membrane via the highly regulated active expulsion of calcium spikes. PMCA (plasma membrane calcium ATPase) isoforms cans also have tissue-specific roles, such as the regulation of the rate of clot retraction in platelets. There are more than 30 splice variants formed from the four PMCA isoforms, each differing in its affinity for calcium and calmodulin, with some isoforms showing tissue-specific expression. PMCA isoforms are differently regulated by protein kinases (PKA, PKC) by proteases (calpain), by effector caspases, and by interaction with phospholipids (phosphotidylserine, phosphatidylinositol), which act to shape the time course of the calcium signals. PMCAs can be distinguished from sarcoplasmic endoplasmic reticulum calcium ATPases by the addition of an extended C-terminal tail that forms an auto-inhibitory domain, providing a mechanism for regulation of PMCA activity by cytosolic calcium concentration. The binding of calmodulin to this domain relieves the inhibition.  

 

The endoplasmic reticulum (ER) plays an important role in regulating cytosolic calcium levels through SERCA pumps, which accumulate calcium in the ER lumen. The mobilization of calcium from intracellular organelles is highly specialized in cardiac and skeletal muscle. In skeletal muscle, calcium ions are transported against a concentration gradient from the cytoplasm into the SR, which causes the relaxation of muscle cells following the excitatory effect of hight cytosolic calcium. In cardiac muscle, the control of intracellular calcium is essential for the regulation of cardiac contractility, and relies upon SERCA and PMCA pumps. SERCA pumps display greater homology with sodium/potassium pumps than with PMCA pumps, most differences occurring in the transmembrane domain. The three human SERCA genes encode up to 10 isoforms by alternative splicing.

 

 

Regulation & Control

 

Calcium ions play a crucial role in the metabolism and physiology of eukaryotes.  Calcium exists as a gradient across the plasma membrane, with extracellular concentrations being about 10,000 times higher than intracellular ones.  Inside the cell, calcium concentrations can vary between different organelles, the transport of calcium between the cytoplasm and organelles such as the sarcoplasmic and endoplasmic reticulum acting to control cytosolic calcium concentrations.  Signaling events often involve an influx of calcium across the plasma membrane, or release of calcium from the sarcoplasmic or endoplasmic reticulum, where the increase in cytosolic calcium can initiate or alter cellular processes.  A whole range of cellular processes is regulated by the free cytosolic calcium concentration, ranging from transcription control and cell survival to neurotransmitter release and muscle function.  In order for a cell to use calcium as a signaling molecule, the cell must create calcium gradients across membranes.  To obtain such concentration differences, calcium ions need to be actively pumped across membranes against a concentration gradient.  Cells use calcium pumps to direct the flow of calcium ions through the plasma membrane or organelle membranes, and the resulting gradients are used in a variety of signalling systems mediated by gated ion channels.  Calcium pumps are ATPases that transport ions across membranes using energy obtained from the hydrolysis of ATP.

 

Every time a muscle is moved, it requires the combined action of trillions of myosin motors.  Muscle cells use Calcium ions to coordinate this massive molecular effort.  When a muscle cell is given the signal to contract from its associated nerves, it releases a flood of calcium ions from the sarcoplasmic reticulum that surrounds the bundles of actin and myosin filaments.  The calcium ions rapidly spread and bind to tropomyosins on the actin filaments. They shift shape slightly and allow myosing to bind and begin climbing up the filament.  These trillions of myosin motors will continue climbing, contracting the muscle, until the calcium is removed.

 

The calcium pump is found in the membrane of the sarcoplasmic reticulum, and allows muscles to relax after the frenzied wave of calcium-induced contraction.  Powered by ATP, the calcium pump pumps calcium ions back into the sarcoplasmic reticulum, reducing the calcium level around the actin and myosin filaments and allowing the muscle to relax.  Calcium ions are also used for signaling inside other cells, and similar pumps are found in the cell membrane of most cells.  They constantly work to reduce the amount of calcium to very low levels, preparing the cell.  Then, at a moment’s notice, the cell can allow a flood of calcium to enter, spreading the signal to all corners.

 

The calcium pump has a large domain poking out on the outside of the sarcoplasmic reticulum, and a region that is embedded in the membrane, forming a tunnel to the other side.  For each ATP broken, it transfers two calcium ions through the membrane, and two or three hydrogen ions back in the opposite direction.  The calcium pump bends and flexes during the pumping cycle and goes through a cycle of changes.

 

Calcium pumps transport Ca+2 from the cytosol to the lumen of the sacroplasmic reticulum. Calcium pumps establish a large concentration gradient of calcium between the lumen and the cytosol.  When calcium channels open, Ca+2 floods out of the sarcoplasmic reticulum which allows muscle contraction.  Calcium pumps transport Ca+2 back into the lumen to facilitate relaxation.