Pumping Calcium

An ornate ion pump manages rapid outflow of calcium.

In the beginning, the egg cell experienced a wave of calcium release, triggered by union with a sperm cell. This blocked other sperm from entering, and prepared the egg to become a zygote and embark on embryogenesis. It is but one example of the pervasive role of calcium signaling among animals. Another is the muscle activation cycle, which relies on calcium release from the specialized sarcoplasmic reticulum (in response to a nerve activation) to get the cell as a whole contracting. Generally, calcium is kept very low in the cytoplasm, and high in the endoplasmic reticulum and outside the cell. Thus, channels gated by electrical activation or other signals can cause rapid cytoplasmic calcium spikes and signal widely within a cell. 

On the flip side, there have to be pumps that keep the cytoplasmic concentration low, and a recent paper elucidates the structure of one such pump that is remarkably fast, while also closely regulated. It is an impressive machine. PMCA2 is an ATP-using calcium pump that sits in the plasma membrane and carries out what is called the Post-Albers cycle. This is a flip-switch mechanism for pumping ions, where ATP drives conformational switches alternately exposing ion binding sites to each side of the membrane. When the pore is open to the cytoplasm, there is no competition from higher concentrations outside, so the active site can bind one internal calcium, given a high-affinity site. Then, after the conformational switch, the pore is exposed to the outside, and at the same time the site is reconfigured to be lower-affinity, releasing the calcium ion into a high concentration environment. Neurons especially use calcium signaling extensively to operate synapses and regulate growth and development. Their rapid and frequent signaling requires a pump that has especially high capacity. PMCA2 operates at a maximal rate of several thousands of Ca2+ ions pumped per second.

Cartoon of the Post-Albers cycle, which is shared by a large family of active ATP-using pumps that transfer ions against their chemical concentration gradient. M is the main transmembrane domain of the pump, where the ions traverse the membrane. The N, P, and A domains are regulatory, especially binding and cleaving ATP  at an interface between the N, P, and A domain. The cycle links power steps (1,2) with conformational changes that carefully gate the pumping process.

And that is not all. Since calcium has a charge of 2+ and this pump does not intend to alter charge across the membrane, the pump simultaneously has binding sites for counter-ions (generally two OH-) that are transferred in the opposite direction from the calcium. Not only that, but every pump of this kind requires regulation of various kinds. PMAC2 is activated by phosphatidyl inositol 4,5 bisphosphate (PIP2), which is another important signaling molecule generated by specific PI kinases in response to activation of G-protein coupled receptors or protein kinase C, which may respond to external signals. In very general terms, these tend to be pro-growth or stress-induced pathways. These regulatory processes can tune the overall rate of recovery from rapid Ca2+ signaling events, by adjusting the level and activity of pumps like PMAC2. 

ATP binds at the N/P/A domain interface, and its hydrolysis (and loss of ADP) generates extensive shape changes, including into the transmembrane M domain. At the very bottom, the calcium ion is shown in green, bound inside the M domain pumping channel. The motions here are subtle, but enough to dramatically reshape the calcium channel.

The authors, using various substrate variants and other tricks, were able to develop structures of PMAC2 in several steps of the pumping cycle, using cryo-electron microscopy. The ATPase site in the N domain (red) is far from the channel that conducts the calcium ion (brown, far bottom). They show extensive shape changes from binding or losing the ATP molecule, though they mostly concern the intracellular domains (red, blue, yellow). The effects on the transmembrane pore domain are rather subtle, shown on right. The authors claim that, compared to other pumps of this large family, the structural changes are significantly less, suggesting that evolution for speed has caused the mechanism to become more efficient, with less wasted motion per conduction event, at least in the channel region itself.

Relation of the PIP2 binding domain (orange/red stick figures) to the calcium core binding site. PIP2 appears to be essential for rapid pump operation. At bottom is shown some schematics of the gating provided by PIP2 in bound and unbound states, especially via the D873 side chain (negatively charged aspartic acid).

They also find that the activating molecule PIP2 is neatly parked right next to the main calcium binding and conduction region, and is more or less essential for enzyme activity. In the graph above (e), they show that several single mutations made in the calcium binding high affinity site, for example switching the negatively charged D873 for the positively charged K (lysine), kills ion pumping activity. Mutation of the PIP2 binding pocket (KKQ->TLL, around position 347) likewise kills enzyme activity.

Relation of the counter-ion channel (red dots) with the calcium channel. Both are essential parts of the mechanism. Closeups with the coordinating protein side chains shown on the right.

The whole mechanism is alluded to in the last figure, where the central calcium binding site is shown, with the general direction of calcium pumping. The counter-ion transport area is shown nearby as a flurry of red dots (standing for water molecules, which at this scale are interchangeable with OH ions). Specific single mutations in either area, either changing negatively charged E412 to positively charged lysine at the calcium binding pocket, or changing polar S877 in the water/hydroxy binding area to the bulky and hydrophobic F (phenylalanine), each kill pumping activity (graph). 

While it would be ideal to have a more dynamic representation of what is going on, the new structures give tremendous detail, including the associated ATP, PIP2, calcium, and water molecules. The mutations also nail down several functional points. Obviously a rather intricate and well-oiled machine that keeps its bit of cellular calcium homeostasis on an even keel. It is hard to believe that the sum of thousands of machines like this one is life, but the deeper we look the more true that appears to be.

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