Studies of Na, K‐ATPase Structure and Function Using Baculovirus

Blanco, Gustavo; Hatfield, Wendy R.; Minor, Nicole T.; Sánchez, Gladis; Koster, Joseph C.; DeTomaso, Anthony W.; Mercer, Robert W.

doi:10.1111/j.1749-6632.1997.tb52228.x

Cited by 7 publications

(7 citation statements)

References 22 publications

(10 reference statements)

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“…It provides the advantage that Sf9 insect cells have no endogenous Na,K-ATPase; therefore, activity of the expressed foreign protein can be studied in an environment free of any contaminating Na,K-ATPase. 42 We infected Sf9 cells with baculoviruses that direct the expression of the Na,K-ATPase α4 and β1 subunits. Whole lysates from the cells were used, and the Na,K-ATPase activity under saturating amounts of Na + , K + , and ATP was measured as previously described.…”

Section: Resultsmentioning

confidence: 99%

Design, Synthesis, and in Vitro and in Vivo Evaluation of Ouabain Analogues as Potent and Selective Na,K-ATPase α4 Isoform Inhibitors for Male Contraception

et al. 2018

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Na,K-ATPase α4 is a testis-specific plasma membrane Na+ and K+ transporter expressed in sperm flagellum. Deletion of Na,K-ATPase α4 in male mice results in complete infertility, making it an attractive target for male contraception. Na,K-ATPase α4 is characterized by a high affinity for the cardiac glycoside ouabain. With the goal of discovering selective inhibitors of the Na,K-ATPase α4 and of sperm function, ouabain derivatives were modified at the glycone (C3) and the lactone (C17) domains. Ouabagenin analogue 25, carrying a benzyltriazole moiety at C17, is a picomolar inhibitor of Na,K-ATPase α4, with an outstanding α4 isoform selectivity profile. Moreover, compound 25 decreased sperm motility in vitro and in vivo and affected sperm membrane potential, intracellular Ca2+, pH, and hypermotility. These results proved that the new ouabagenin triazole analogue is an effective and selective inhibitor of Na,K-ATPase α4 and sperm function.

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Section: Resultsmentioning

confidence: 99%

Design, Synthesis, and in Vitro and in Vivo Evaluation of Ouabain Analogues as Potent and Selective Na,K-ATPase α4 Isoform Inhibitors for Male Contraception

et al. 2018

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“…Above all, the insect cell system permits the functional expression of multimeric membrane proteins. Interactions between the α and β subunits of Na,K-ATPase, and the kinetic properties of Na,K-ATPase isoenzymes, have been studied in insect cells, with heterodimer function measured by ouabain-sensitive ATPase activity (reviewed in [32]). In addition, the expression of wildtype [22,33,34] and mutated [29] rat gastric H,K-ATPase has been studied in insect cells, with the functional capacity of HKαβ heterodimers being assessed by SCH 28080-sensitive phosphorylation of the HKα.…”

Section: Discussionmentioning

confidence: 99%

H,K-ATPase α subunit C-terminal membrane topology: epitope tags in the insect cell expression system

Smolka¹,

Larsen

Schweinfest

et al. 1999

Biochemical Journal

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The H,K-ATPase responsible for gastric acidification is a heterodimeric (alpha and beta subunit) P-type ATPase, an integral protein of parietal cell apical membranes, which promotes the electroneutral exchange of K+ for protons, is stimulated by K+ and is inhibited by 2-methyl-8-(phenylmethoxy)imidazo[1, 2-alpha]pyridine-3-acetonitrile (SCH 28080). Hydropathy analysis of the catalytic alpha subunit has been interpreted in terms of four N-terminal transmembrane domains, a cytoplasmically oriented segment containing ATP binding and phosphorylation sites, and a C-terminal region with four or six putative transmembrane domains. Several lines of evidence implicate the C-terminal region of P-type ATPases in cation-binding and occlusion, conformational changes, and interactions with the beta subunit (HKbeta), making the definition of topology a prerequisite for understanding the structural basis of these functions. Influenza haemagglutinin epitopes (YPYDVPDYA; flu tag) were inserted in predicted hydrophilic segments of the alpha subunit (HKalpha) to establish the membrane orientation of two amino acids with different predicted topologies in the C-terminal four- and six-transmembrane models. Wild-type and mutated HKalpha and HKbeta cDNA species were expressed in insect cells (Sf9) via recombinant baculovirus infection, and expression of H,K-ATPase was verified by immunoblotting with HKalpha- and HKbeta-specific and flu-tag-specific antibodies. Functional assays showed K+-stimulated, SCH 28080-sensitive ATPase activity, confirming neo-native topology in H,K-ATPase heterodimers expressed in Sf9 cells. The topology of flu tags was determined by microsomal protease protection assays in Sf9 cells and immunolabelling of HKalpha and HKbeta in intact and permeabilized Sf9 cells. In addition, MS of native H,K-ATPase tryptic peptides identified cytoplasmically oriented HKalpha residues. The results indicated cytoplasmic exposure of Leu844 and Phe996, and luminal exposure of Pro898, leading to a revised secondary structure model of the C-terminal third of HKalpha.

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“…However, the only evidence of a mechanism exists with studies involving the Na+ /K+ -ATPase inhibition (Ahmed and Thomas, 1971;Morohashi et al, 1991). Unfortunately the role of these enzymes is poorly understood in insects mainly because they normally do not appear to have any activity associated with them (Blanco, 1997).…”

Section: Discussionmentioning

confidence: 99%

“…Perhaps linoleic acid is used more as an antibacterial agent as (3) inhibited more gram-positive bacteria than did (2). Unfortunately, the exact mechanism of Na+ /K+ -ATPase in insects is not well documented as many of these enzymes are found to be present but with little to no activity (Blanco, 1997). Research done by Gatto et al (2001) does suggest that Na+ /K+ -ATPases in insects may play roles in transport for intracellular organelles because fully functional enzymes were discovered in such locations.…”

Section: Potential Roles Of Unsaturated Fatty Acids In Cyanobacteriamentioning

confidence: 99%

Mosquito Larvicides from Cyanobacteria

Berry¹

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Studies of Na, K‐ATPase Structure and Function Using Baculovirus

Cited by 7 publications

References 22 publications

Design, Synthesis, and in Vitro and in Vivo Evaluation of Ouabain Analogues as Potent and Selective Na,K-ATPase α4 Isoform Inhibitors for Male Contraception

Design, Synthesis, and in Vitro and in Vivo Evaluation of Ouabain Analogues as Potent and Selective Na,K-ATPase α4 Isoform Inhibitors for Male Contraception

H,K-ATPase α subunit C-terminal membrane topology: epitope tags in the insect cell expression system

Mosquito Larvicides from Cyanobacteria

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