Structure of the 5th transmembrane segment of the Na,K‐ATPase α subunit: a cysteine‐scanning mutagenesis study

Abstract

To study the structure of the pathway of cations across the Na,K-ATPase, we applied the substituted cysteine accessibility method to the putative 5th transmembrane segment of the K K subunit of the Na,K-ATPase of the toad Bufo marinus. Only the most extracellular amino acid position (A 796 ) was accessible from the extracellular side in the native Na,K-pump. After treatment with palytoxin, six other positions (Y 778 , L 780 , S 782 , P 785 , E 786 and L 791 ), distributed along the whole length of the segment, became readily accessible to a small-size methanethiosulfonate compound (2-aminoethyl methanethiosulfonate). The accessible residues are not located on the same side of an K K-helical model but the pattern of reactivity would rather suggest a L L-sheet structure for the inner half of the putative transmembrane segment. These results demonstrate the contribution of the 5th transmembrane segment to the palytoxininduced channel and indicate which amino acid positions are exposed to the pore of this channel. ß

Electrogenicity of Na,K- and H,K-ATPase Activity and Presence of a Positively Charged Amino Acid in the Fifth Transmembrane Segment

“…Because of the large difference in Na,K-pump transport activity at 5 and 40 mM K ϩ observed in mutant S782A, our results first confirm earlier studies showing that this residue has a large influence on the apparent K ϩ affinity of the Na,K-pump (21-23); more precisely, the S775A mutation of the sheep Na,K-ATPase yields a very low affinity for K ϩ without changes in the voltage dependence of this affinity (21). This residue is closely associated with the cation pathway through the Na,K-pump as recently shown by its accessibility to water-soluble sulfhydryl reagents when the Na,K-ATPase has been modified to a channel by palytoxin (34).…”

The Fourth Transmembrane Segment of the Na,K-ATPase α Subunit

“…Seven nanograms of ␣ subunit and 1 ng of ␤ subunit cRNA were mixed and co-injected in a total volume of 50 nl into stage V-VI X. laevis oocytes (13). The injected oocytes were incubated for 3-5 days in a modified Barth's solution and loaded with Na ϩ by exposure to a K ϩ -free solution overnight before the electrophysiological measurements as described earlier (8).…”

“…We have recently published the results of studies in which we have examined the accessibility of the residues of the fifth and sixth TM segments by systematic mutagenesis of all the residues of these domains into cysteine (8,9) and then probing the accessibility of the introduced thiol groups with sulfhydryl reagents. These studies have allowed us to define the aspects of the corresponding TM helices that form the pathway for cations across the membrane.…”

Actin cytoskeleton of rabbit intestinal cells is a target for potent marine phycotoxins

“…In the same way, palytoxin, one of the most potent marine neurotoxins, produces an intoxication named clupeotoxism, whose symptomatology is similar to ciguatera although more serious and with a high fatality rate (Onuma et al, 2000). Recent works suggests that this toxin shows specificity for Na + /K + pumps in Xenopus oocyte (Wang and Horisberger, 1997;Guennoun and Horisberger, 2000), but there are no data on its biological activity in a cellular intestinal model. By contrast, ostreocin-D is a structural analogue of palytoxin, whose mechanism of action and toxicological effects have not yet been elucidated, although seafood contamination with ostreocin-D is becoming an increasing problem in some Mediterranean countries.…”