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The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic Ca(2+), initiated by the release of Ca(2+) from the sarcoplasmic reticulum and terminated by re-sequestration of Ca(2+) into the sarcoplasmic reticulum as the main mechanism of Ca(2+) removal. Re-sequestration requires active transport and is catalysed by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), which has a key role in defining the contractile properties of skeletal and heart muscle tissue. The activity of SERCA is regulated by two small, homologous membrane proteins called phospholamban (PLB, also known as PLN) and sarcolipin (SLN). Detailed structural information explaining this regulatory mechanism has been lacking, and the structural features defining the pathway through which cytoplasmic Ca(2+) enters the intramembranous binding sites of SERCA have remained unknown. Here we report the crystal structure of rabbit SERCA1a (also known as ATP2A1) in complex with SLN at 3.1 Å resolution. The regulatory SLN traps the Ca(2+)-ATPase in a previously undescribed E1 state, with exposure of the Ca(2+) sites through an open cytoplasmic pathway stabilized by Mg(2+). The structure suggests a mechanism for selective Ca(2+) loading and activation of SERCA, and provides new insight into how SLN and PLB inhibition arises from stabilization of this E1 intermediate state without bound Ca(2+). These findings may prove useful in studying how autoinhibitory domains of other ion pumps modulate transport across biological membranes.

The Na+/K+-ATPase enzyme pumps three sodium ions out of the cell and two potassium ions into the cell while splitting a single molecule of ATP. This process generates the chemical gradients across the plasma membrane that are essential to electrical excitation and ion balance in animal cells. Here the authors determine that the C-terminus of the ATPase is a key regulator of a previously unrecognized ion pathway. Their data suggest that a cytoplasmic proton can enter and stabilize site III when empty (in the potassium-bound state); when the potassium ion is released, the proton returns to the cytoplasm, thus permitting an overall asymmetric stoichiometry of the transported ions. The C-terminus is crucial for pump function as demonstrated by reports that at least eight mutations in the region cause severe neurological diseases. The Na+/K+-ATPase pumps three sodium ions out of and two potassium ions into the cell while splitting a single molecule of ATP. Here it is found that the carboxy terminus of the ATPase's α-subunit is also a key regulator of a previously unrecognized ion pathway. The data indicate that, in the ATPase's potassium-bound state, a cytoplasmic proton can enter and stabilize site III when empty. When potassium is released, the proton returns to the cytoplasm, thus permitting an overall asymmetric stoichiometry of the transported ions. The Na+/K+-ATPase pumps three sodium ions out of and two potassium ions into the cell for each ATP molecule that is split, thereby generating the chemical and electrical gradients across the plasma membrane that are essential in, for example, signalling, secondary transport and volume regulation in animal cells. Crystal structures of the potassium-bound form of the pump revealed an intimate docking of the α-subunit carboxy terminus at the transmembrane domain1,2. Here we show that this element is a key regulator of a previously unrecognized ion pathway. Current models of P-type ATPases operate with a single ion conduit through the pump3,4,5, but our data suggest an additional pathway in the Na+/K+-ATPase between the ion-binding sites and the cytoplasm. The C-terminal pathway allows a cytoplasmic proton to enter and stabilize site III when empty in the potassium-bound state, and when potassium is released the proton will also return to the cytoplasm, thus allowing an overall asymmetric stoichiometry of the transported ions. The C terminus controls the gate to the pathway. Its structure is crucial for pump function, as demonstrated by at least eight mutations in the region that cause severe neurological diseases6,7. This novel model for ion transport by the Na+/K+-ATPase is established by electrophysiological studies of C-terminal mutations in familial hemiplegic migraine 2 (FHM2) and is further substantiated by molecular dynamics simulations. A similar ion regulation is likely to apply to the H+/K+-ATPase and the Ca2+-ATPase.