Sampedro Castañeda M , Zanoteli E , Scalco RS , Scaramuzzi V , Marques Caldas V , Conti Reed U , da Silva AMS , O'Callaghan B , Phadke R , Bugiardini E , Sud R , McCall S , Hanna MG , Poulsen H , Männikkö R , Matthews E .

MR/K000608/1 Medical Research Council , MR/M006948/1 Medical Research Council , Wellcome Trust , 209583/Z/17/Z Wellcome Trust

             sodium channel activity

           HYPOKALEMIC PERIODIC PARALYSIS, TYPE 2; HOKPP2

	Figure 1. Muscle pathology and genetic analysis of the hypoPP patient. (A) Histological examination of a biceps brachii muscle biopsy performed at age 2 years and 3 months. (i) Haematoxylin and eosin staining showed variation in fibre diameter, increased internal nuclei (black arrows), a necrotic fibre (white arrow) and subtle endomysial fibrosis (also seen in iii) (ii) Myosin ATPase histochemistry at pH 9.4 indicated type I fibre predominance (pale stained fibres). (iii) Gomori trichrome staining. (B) Sequence chromatogram demonstrating the heterozygous mutation c.2336 G>A; p.S779N in the proband (above) and sequence alignment of human Na+/K+-ATPase alpha subunits. ATP1A2-S779 and analogous serine residues are underlined. (C) Structural context of S779. Left: An overview of the Na+/K+-ATPase structure with the alpha subunit in grey, the beta subunit in blue and the gamma subunit in red. The two potassium ions are yellow spheres, S779 is in orange stick, and the ion-coordinating transmembrane helices are light cyan (M4), wheat (M5) and light blue (M6). The boundaries of the membrane are indicated by horizontal lines. Right: Close-ups of the ion binding sites viewed from the extracellular side, top with two potassium ions (yellow), bottom with three sodium ions (violet). S779 is close to ion binding sites I and III. The figure was made using PDB structures 2ZXE (potassium bound) and 3WGU (sodium bound).
	Figure 2. Steady state and transient Na+ currents of WT and p.S779N pumps in the absence of extracellular K+. (A) Representative raw current traces in absence (left) and presence (middle) of 10 mM ouabain and the ouabain-sensitive currents (right) in wild-type (black) and p.S779N (red) pumps in response to voltage steps from −160 to +60 mV, in 20-mV increments. Steady state currents at each voltage were measured in the last 50 ms of the 200 ms stimulus, indicated by dotted lines. (B) Average ouabain-sensitive steady state leak currents. Slope conductance between −140 mV and 0 mV was 0.75 ± 0.08 nA/mV for the p.S779N pump (n = 27) while it was close to 0 for the wild-type pump (n = 29). (C) Charge-voltage relationships. Na+ charge transfer was determined from the integral of the first 50 ms of the current trace at −30 mV following the steps to test voltages. Individual QV curves were fit by a standard Boltzmann function and normalized to their respective fits. V1/2: p.S779N −95.4 ± 7.1 mV, n = 25; wild-type 2.6 ± 1.7 mV, n = 29; slope: p.S779N 66.8 ± 4.8 mV, wild-type 28.9 ± 0.7 mV, P < 10−5, unpaired t-test. (D) Rate constants of transient currents at the onset of the stimulus. Solid line represents a Boltzmann fit and yielded the overall forward/backward rate constants (p.S779N, 773.9 ± 27.7/542.6 ± 12.7 s−1, n = 22 wild-type 102.3 ± 3.6/882.2 ± 32.9 s−1, n = 26; P < 0.0001, unpaired t-test).
	Figure 3. Forward pumping currents of WT and p.S779N α 2 at various extracellular [K+]o. (A) Example current traces of wild-type (black) and mutant (red) pumps in response to 200 ms steps from −160 to +40 mV, from a holding voltage of −30 mV in absence (left) and presence (middle) of 15 mM K+, and in presence of 15 mM K+ and 10 mM ouabain (right). K+-induced pump currents were isolated off-line by subtraction of currents in the presence of ouabain 10 mM. (B) Current-voltage relationship of steady state ouabain-sensitive currents measured in last 50 ms of each step (p.S779N n = 17; wild-type n = 20; P < 0.0001 two-way ANOVA). (C) Current-voltage relationship of ouabain-sensitive currents normalized to total Na+ charge transfer (Qtot) in absence of K+, as a measure of functional protein expression (p.S779N n = 7; wild-type n = 13; P = 0.001, two-way ANOVA). Qtot was estimated in a subset of cells where lower amplifier gain was used to minimize current saturation because of capacitive artefacts. Ten traces were averaged to reduce the noise and then used to determine the span of the Boltzman curve (Qtot: S779N 5.4 ± 0.9 nC, n = 7; wild-type 5.5 ± 0.7 nC, n = 13). (D) Extracellular apparent K+ affinity of the ATPases: current-voltage relationships of wild-type (i) and mutant pumps (ii) in various [K+]o conditions normalized to the outward current at +20 mV in 15 mM K+ (p.S779N n = 5–6; wild-type n = 3–5). (E) Concentration-response curves obtained from the data in D at three representative voltages. Dotted lines represent fits to a modified Hill equation (wild-type EC50 at −100, −60, 0 mV: 3.5 ± 0.3, 2.2 ± 0.2, 1.3 ± 0.1 mM, n = 5; P < 0.0001 repeated measures ANOVA; p.S779N EC50 at −100, −60, 0 mV: 6.9 ± 0.4, 6.9 ± 0.2, 7.5 ± 0.7 mM, n = 5; P = 0.7). Curves obtained from separate experiments were normalized to the top and bottom values of the fit and averaged across oocytes. Hill coefficients were voltage independent and did not change significantly between pump variants: wild-type 1.54 ± 0.06, S779N 1.56 ± 0.06 (P = 0.3, two-way ANOVA across the voltages). (F) Overall voltage dependence of apparent [K+] affinity (wild-type n = 3–5; p.S779N n = –5, P < 0.001 two-way ANOVA).
	Figure 4. Ionic contributions to the p.S779N ATPase leak current. (A) Representative current traces in 0 [K+]o for wild-type (black) and p.S779N (red) illustrating changes in leak currents with varying [H+]o. (B) Steady state current-voltage relationships in different pH conditions normalized to current at +20 mV in pH 7.4 (n = 4 for both). Linear fits to raw leak currents (not shown) in p.S779N indicate an increase in slope conductance with acidification from 0.54 ± 0.1 to 0.85 ± 0.2 nA/mV (P = 0.03, paired t-test) and a shift in Erev from −12.7 ± 2.5 to −2.2 ± 2.6 mV (P = 0.01). For pH 8.2 the slope was 0.43 ± 0.1 (P = 0.15) and Erev = −17.8 ± 2.2 mV (P = 0.06). (C) Increase in inward leak currents in p.S779N pump with acidification in the presence of 15 mM [K+]o; comparison with linear leak in Na+-only (pH 7.4) conditions (n = 4). Currents have been normalized to amplitude at +20 in pH 7.4. (D) Average steady state currents for wild-type (left) or p. S779N (right) pumps in control conditions (115 mM Na+, 0 K+) and when 86 mM Na+ is substituted with NMDG+. Currents have been normalized to control value at +20 mV (wild-type n = 6, P = 0.008; p.S779N n = 7, P = 0.001 repeated measures ANOVA). For the mutant pump, Erev shifted from −16.2 ± 1.5 mV in Na+ to −48.8 ± 10 mV in Na/NMDG+ (n = 7, P = 0.016, Wilcoxon paired t-test).

Argüello, Substitutions of serine 775 in the alpha subunit of the Na,K-ATPase selectively disrupt K+ high affinity activation without affecting Na+ interaction. 1995, Pubmed

Argüello, Substitutions of serine 775 in the alpha subunit of the Na,K-ATPase selectively disrupt K+ high affinity activation without affecting Na+ interaction. 1995, Pubmed
Bassi, A novel mutation in the ATP1A2 gene causes alternating hemiplegia of childhood. 2004, Pubmed
Berry, Differential distribution and regulation of mouse cardiac Na+/K+-ATPase alpha1 and alpha2 subunits in T-tubule and surface sarcolemmal membranes. 2007, Pubmed
Blostein, Evidence that Ser775 in the alpha subunit of the Na,K-ATPase is a residue in the cation binding pocket. 1997, Pubmed
Bøttger, Migraine- and dystonia-related disease-mutations of Na+/K+-ATPases: relevance of behavioral studies in mice to disease symptoms and neurological manifestations in humans. 2012, Pubmed
Cannon, Channelopathies of skeletal muscle excitability. 2015, Pubmed
Clausen, The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease. 2017, Pubmed
DiFranco, Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+. 2015, Pubmed
Fong, Intracellular sodium-activity at rest and after tetanic stimulation in muscles of normal and dystrophic (dy2J/dy2J) C57BL/6J mice. 1986, Pubmed
Golovina, Regulation of Ca2+ signaling by Na+ pump alpha-2 subunit expression. 2003, Pubmed
Han, Extracellular potassium dependence of the Na+-K+-ATPase in cardiac myocytes: isoform specificity and effect of phospholemman. 2009, Pubmed
Hilbers, Tuning of the Na,K-ATPase by the beta subunit. 2016, Pubmed , Xenbase
Horisberger, Functional differences between alpha subunit isoforms of the rat Na,K-ATPase expressed in Xenopus oocytes. 2002, Pubmed , Xenbase
Illarionova, Role of Na,K-ATPase α1 and α2 isoforms in the support of astrocyte glutamate uptake. 2014, Pubmed
Isaksen, Hypothermia-induced dystonia and abnormal cerebellar activity in a mouse model with a single disease-mutation in the sodium-potassium pump. 2017, Pubmed , Xenbase
Juhaszova, Na+ pump low and high ouabain affinity alpha subunit isoforms are differently distributed in cells. 1997, Pubmed
Jurkat-Rott, K+-dependent paradoxical membrane depolarization and Na+ overload, major and reversible contributors to weakness by ion channel leaks. 2009, Pubmed
Larsen, Contributions of the Na⁺/K⁺-ATPase, NKCC1, and Kir4.1 to hippocampal K⁺ clearance and volume responses. 2014, Pubmed , Xenbase
Li, The third sodium binding site of Na,K-ATPase is functionally linked to acidic pH-activated inward current. 2006, Pubmed , Xenbase
Matthews, Voltage sensor charge loss accounts for most cases of hypokalemic periodic paralysis. 2009, Pubmed
Matthews, Acetazolamide efficacy in hypokalemic periodic paralysis and the predictive role of genotype. 2011, Pubmed
Meier, Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase {alpha} subunit. 2010, Pubmed , Xenbase
Meyer, On the effect of hyperaldosteronism-inducing mutations in Na/K pumps. 2017, Pubmed , Xenbase
Mitchell, Sodium and proton effects on inward proton transport through Na/K pumps. 2014, Pubmed , Xenbase
Morth, Crystal structure of the sodium-potassium pump. 2007, Pubmed
Nyblom, Crystal structure of Na+, K(+)-ATPase in the Na(+)-bound state. 2013, Pubmed
Orlowski, Tissue-specific and developmental regulation of rat Na,K-ATPase catalytic alpha isoform and beta subunit mRNAs. 1988, Pubmed
Pedersen, Contribution to Tl+, K+, and Na+ binding of Asn776, Ser775, Thr774, Thr772, and Tyr771 in cytoplasmic part of fifth transmembrane segment in alpha-subunit of renal Na,K-ATPase. 1998, Pubmed
Pelzer, Recurrent coma and fever in familial hemiplegic migraine type 2. A prospective 15-year follow-up of a large family with a novel ATP1A2 mutation. 2017, Pubmed
Plaster, Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome. 2001, Pubmed , Xenbase
Poulsen, Neurological disease mutations compromise a C-terminal ion pathway in the Na(+)/K(+)-ATPase. 2010, Pubmed , Xenbase
Price, Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme. 1988, Pubmed
Radzyukevich, Tissue-specific role of the Na,K-ATPase α2 isozyme in skeletal muscle. 2013, Pubmed
Sokolov, Gating pore current in an inherited ion channelopathy. 2007, Pubmed , Xenbase
Stanley, Intracellular Requirements for Passive Proton Transport through the Na+,K+-ATPase. 2016, Pubmed , Xenbase
Stanley, Importance of the Voltage Dependence of Cardiac Na/K ATPase Isozymes. 2015, Pubmed , Xenbase
Struyk, A Na+ channel mutation linked to hypokalemic periodic paralysis exposes a proton-selective gating pore. 2007, Pubmed , Xenbase
Suetterlin, Muscle channelopathies: recent advances in genetics, pathophysiology and therapy. 2014, Pubmed
Vasilyev, Effect of extracellular pH on presteady-state and steady-state current mediated by the Na+/K+ pump. 2004, Pubmed , Xenbase
Vedovato, Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps. 2014, Pubmed , Xenbase
Wang, A conformation of Na(+)-K+ pump is permeable to proton. 1995, Pubmed , Xenbase
Wu, A sodium channel knockin mutant (NaV1.4-R669H) mouse model of hypokalemic periodic paralysis. 2011, Pubmed