A bimodular mechanism of calcium control in eukaryotes

Abstract

Calcium ions (Ca(2+)) have an important role as secondary messengers in numerous signal transduction processes, and cells invest much energy in controlling and maintaining a steep gradient between intracellular (∼0.1-micromolar) and extracellular (∼2-millimolar) Ca(2+) concentrations. Calmodulin-stimulated calcium pumps, which include the plasma-membrane Ca(2+)-ATPases (PMCAs), are key regulators of intracellular Ca(2+) in eukaryotes. They contain a unique amino- or carboxy-terminal regulatory domain responsible for autoinhibition, and binding of calcium-loaded calmodulin to this domain releases autoinhibition and activates the pump. However, the structural basis for the activation mechanism is unknown and a key remaining question is how calmodulin-mediated PMCA regulation can cover both basal Ca(2+) levels in the nanomolar range as well as micromolar-range Ca(2+) transients generated by cell stimulation. Here we present an integrated study combining the determination of the high-resolution crystal structure of a PMCA regulatory-domain/calmodulin complex with in vivo characterization and biochemical, biophysical and bioinformatics data that provide mechanistic insights into a two-step PMCA activation mechanism mediated by calcium-loaded calmodulin. The structure shows the entire PMCA regulatory domain and reveals an unexpected 2:1 stoichiometry with two calcium-loaded calmodulin molecules binding to different sites on a long helix. A multifaceted characterization of the role of both sites leads to a general structural model for calmodulin-mediated regulation of PMCAs that allows stringent, highly responsive control of intracellular calcium in eukaryotes, making it possible to maintain a stable, basal level at a threshold Ca(2+) concentration, where steep activation occurs.

A channelopathy mechanism revealed by direct calmodulin activation of TrpV4

“…Our model is akin to the classical activation mechanism whereby an autoinhibitory interaction is disrupted by the binding of diffusible Ca 2+ -CaM to activate many enzymes (17,18) and some channels (19,20). This model is similar to that proposed by Strotmann et al (10), where the CBD itself binds an N-terminal region (S117-S134) in the absence of CaM.…”

Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains

“…Similar abrupt activation kinetics in response to an increase in modulator concentration have been reported for the autoinhibited Ca 2ϩ -ATPase ACA8 (48). This P-type ATPase is activated by calmodulin binding to two autoinhibitory regions in response to an increase in Ca 2ϩ concentration.…”

“…This P-type ATPase is activated by calmodulin binding to two autoinhibitory regions in response to an increase in Ca 2ϩ concentration. Mathematical modeling of Ca 2ϩ /calmodulin action on ACA8 demonstrates that when two intramolecular autoinhibitors are present, rather than just one, activation becomes almost instantaneous above a certain threshold value of Ca 2ϩ (48). In accordance with this model, a concerted action between several autoinhibitory regions (the N terminus and Regions I and II of the C-terminal domain), which is neutralized by lysophospholipids, could explain why lipid activation of the PM H ϩ -ATPase AHA2 by lysophospholipids is instantaneous above a certain threshold value.…”

Insight into the Family of Na+/Ca2+ Exchangers of Caenorhabditis elegans

“…Cells accomplish this primarily by extruding calcium through plasma membraneembedded plasma membrane Ca 2+ ATPase (PMCA) pumps and utilizing exchanger ion transporters. PMCA proteins are high-affinity/low-capacity pumps that maintain calcium homeostasis over sustained periods of time by removing one Ca 2+ ion for every ATP hydrolyzed (Tidow et al 2012). Exchangers such as Na + /Ca 2+ exchangers (NCX), Na + /Ca 2+ /K + exchangers (NCKX), and calcium/cation exchangers (CCX) are low-affinity/high-capacity ion transporters that rapidly expel calcium ions (Philipson and Nicoll 2000;Philipson et al 2002;Lytton 2007;Nicoll et al 2013).…”