Cation Stoichiometry and Cation Pathway in the Na,K‐ATPase and Nongastric H,K‐ATPase

Abstract

The mechanism of cation translocation by the Na,K-ATPase was investigated by cysteine scanning mutagenesis and measurements of accessibility through exposure to cysteine reagents. In the native protein, accessible residues were found only at the most extracellular residues of the 5th and 6th transmembrane segments (TMS) and the short loop between them. However, after modification by palytoxin a number of residues became accessible along the whole length of the 5th TMS and in the outer half of the 6th TMS, showing the contribution of each of these segments to the "channel" formed by the palytoxin-transformed Na,K-pump. Assuming that this structure is similar in the native and the palytoxin-transformed pump, our data allow us to determine the residues lining the cation pathway from the extracellular solution to their binding sites. A critical position in the 5th TMS contains a lysine conserved in all known nonelectrogenic H,K-ATPases, and a serine in all known electrogenic Na,K-ATPase sequences. Wild-type or mutant Na,K-or H,K-ATPase a subunits were coinjected with the Bufo beta2 subunit in Xenopus oocytes and Rb(86) uptake and electrophysiological measurements were performed. An electrogenic activity was recorded for the H,K-ATPase mutants in which the positively charged lysine had been replaced by neutral or negatively charged residues, while nonelectrogenic transport was observed with the S(782)R mutant of the Na,K-ATPase. The presence or the absence of a positively charged residue at the S(782) position appears to be critical for the stoichiometry of cation exchange.

The role of the third extracellular loop of the Na+,K+‐ATPase α subunit in a luminal gating mechanism

“…The cation entry sites, and the pathway followed by the cations en route from the extracellular solution to their binding sites, have not yet been completely mapped. Cysteine scanning mutagenesis studies of the TM4, TM5 and TM6 helices (Guennoun & Horisberger, 2000, 2002; Horisberger et al 2003) have provided evidence compatible with the model obtained by homology, and support a role of these three helices, and in particular a role for a phenylalanine residue in TM4 (Horisberger et al 2004), but the cation pathway remains poorly defined. Toyoshima et al (2004), in their recent publication of a structure of SERCA in a conformation state similar to the E2P state, have proposed a luminal exit pathway for Ca 2+ between the TM4 and TM6 helices.…”

Measuring Cation Transport by Na,K- and H,K-ATPase in <em>Xenopus</em> Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

“…It is a safe and reliable alternative to radioactive tracer flux assays, and allows addressing a large scope of experimental questions within considerably short time. In accordance, only very limited data have been acquired with radiotracers [24][25][26][27] . The AAS method is particularly useful for H + ,K + -ATPase, which -due to its electroneutral transport activity -cannot be analyzed by standard electrophysiology.…”

Palytoxin causes nonoxidative necrotic damage to PC12 cells in culture

“…It is therefore necessary to investigate the properties and mechanisms of their toxic actions to establish methods of the detoxification and treatment for poisoning. Previously, palytoxin (PTX), a potent marine toxin isolated from the soft coral Palythoa toxica, has been reported to interact with plasma membranes, resulting in the modulation of cation transport across the membranes (Horisberger et al, 2003;Lauffer et al, 1985;Tosteson et al, 1991Tosteson et al, , 1995Tosteson et al, , 2003, thereby causing fatal damage to various cells and tissues through an alteration in the cytoplasmic calcium concentration (Satoh et al, 2003). For instance, this toxin has been shown to primarily impair the myocardium, and has also been reported to cause vasoconstriction in heart and lungs.…”