The Na+,K+-ATPase generates electrochemical gradients for sodium and potassium that are vital to animal cells, exchanging three sodium ions for two potassium ions across the plasma membrane during each cycle of ATP hydrolysis. Here we present the X-ray crystal structure at 3.5 A resolution of the pig renal Na+,K+-ATPase with two rubidium ions bound (as potassium congeners) in an occluded state in the transmembrane part of the alpha-subunit. Several of the residues forming the cavity for rubidium/potassium occlusion in the Na+,K+-ATPase are homologous to those binding calcium in the Ca2+-ATPase of sarco(endo)plasmic reticulum. The beta- and gamma-subunits specific to the Na+,K+-ATPase are associated with transmembrane helices alphaM7/alphaM10 and alphaM9, respectively. The gamma-subunit corresponds to a fragment of the V-type ATPase c subunit. The carboxy terminus of the alpha-subunit is contained within a pocket between transmembrane helices and seems to be a novel regulatory element controlling sodium affinity, possibly influenced by the membrane potential.

Purpose A novel dissolution dynamic nuclear polarization (dDNP) polarizer platform is presented. The polarizer meets a number of key requirements for in vitro, preclinical, and clinical applications. Method It uses no liquid cryogens, operates in continuous mode, accommodates a wide range of sample sizes up to and including those required for human studies, and is fully automated. Results It offers a wide operational window both in terms of magnetic field, up to 10.1 T, and temperature, from room temperature down to 1.3 K. The polarizer delivers a 13C liquid state polarization for [1‐13C]pyruvate of 70%. The build‐up time constant in the solid state is approximately 1200 s (20 minutes), allowing a sample throughput of at least one sample per hour including sample loading and dissolution. Conclusion We confirm the previously reported strong field dependence in the range 3.35 to 6.7 T, but see no further increase in polarization when increasing the magnetic field strength to 10.1 T for [1‐13C]pyruvate and trityl. Using a custom dry magnet, cold head and recondensing, closed‐cycle cooling system, combined with a modular DNP probe, and automation and fluid handling systems, we have designed a unique dDNP system with unrivalled flexibility and performance.

In a mouse mutagenesis screen, we isolated a mutant, Myshkin (Myk), with autosomal dominant complex partial and secondarily generalized seizures, a greatly reduced threshold for hippocampal seizures in vitro, posttetanic hyperexcitability of the CA3-CA1 hippocampal pathway, and neuronal degeneration in the hippocampus. Positional cloning and functional analysis revealed that Myk/؉ mice carry a mutation (I810N) which renders the normally expressed Na ؉ ,K ؉ -ATPase ␣3 isoform inactive. Total Na ؉ ,K ؉ -ATPase activity was reduced by 42% in Myk/؉ brain. The epilepsy in Myk/؉ mice and in vitro hyperexcitability could be prevented by delivery of additional copies of wild-type Na ؉ ,K ؉ -ATPase ␣3 by transgenesis, which also rescued Na ؉ ,K ؉ -ATPase activity. Our findings reveal the functional significance of the Na ؉ ,K ؉ -ATPase ␣3 isoform in the control of epileptiform activity and seizure behavior.alpha3 Na ϩ ,K ϩ ATPase ͉ BAC rescue ͉ epilepsy ͉ forward genetic screen ͉ mouse

Endogenous period is extended in Myk/ + due to longer periods of (I) activity (α). All data are presented as means ± SEM, *P < 0.05, **P < 0.01; ***P < 0.001 compared with +/+ mice.