title: Activity of the Na,K-ATPase α4 isoform is regulated during sperm capacitation to support sperm motility.
abstract: The α4 polypeptide is a testis-specific isoform of the catalytic  subunit of the Na,K-ATPase, which is essential for sperm motility and  fertility. In the present study, we have investigated the regulation of  activity of the α4 isoform and the relevance of this event for sperm  capacitation. We have performed this by taking advantage of the selective high  affinity of α4 for the inhibitor ouabain. Our results show that  ouabain-sensitive hydrolysis of ATP and uptake of (86)Rb,  corresponding to the enzymatic and ion transport activities of α4,  respectively, increased during sperm capacitation in a time-dependent manner.  Specific labeling of α4 with the fluorescent indicator bodipy-ouabain  and immunoblot analysis of biotinylated and streptavidin-precipitated sperm  plasma membrane proteins indicated a capacitation- and time-dependent rise in  levels of active α4 isoform at the sperm surface. Ouabain inhibition of  α4 blocked the increase in total sperm motility and the hyperactive  motility pattern characteristic of sperm capacitation. Moreover, interference  of α4 activity with ouabain partially prevented the intracellular  decrease in Na(+) and the plasma membrane hyperpolarization that  typically accompany sperm capacitation. In contrast, ouabain inhibition of  α4 did not affect the spontaneous sperm acrosomal reaction following  capacitation. Together, these results demonstrate that Na,K-ATPase α4  activity is up-regulated during sperm capacitation through mechanisms that  involve both increases in molecular activity and levels of α4 at the  sperm plasma membrane. This increase in α4 activity helps maintain the  changes in motility that are associated with sperm capacitation, emphasizing  the biologic relevance of the Na,K-ATPase α4 isoform in sustaining sperm  function.
authors.affiliation: Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Blvd, Kansas City, KS 66160, USA.
authors.forename: Tamara
authors.initials: T
authors.lastname: Jimenez
authors.forename: Gladis
authors.initials: G
authors.lastname: Sánchez
authors.forename: Gustavo
authors.initials: G
authors.lastname: Blanco
mesh_terms: D020101:Acrosome Reaction; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006868:Hydrolysis; D008297:Male; D008564:Membrane Potentials; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012414:Rubidium Radioisotopes; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013075:Sperm Capacitation / Q000187:drug effects; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000201:enzymology*; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D004791:Enzyme Inhibitors; D012414:Rubidium Radioisotopes; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; C485720:Atp1a4 protein, rat; D000254:Sodium-Potassium-Exchanging ATPase