title: Intracellular Requirements for Passive Proton Transport through the Na+,K+-ATPase.
abstract: The Na+,K+-ATPase (NKA or Na/K pump) hydrolyzes one ATP to exchange three intracellular Na+ (Na+i) for two extracellular K+ (K+o) across the plasma membrane by cycling through a set of reversible transitions between phosphorylated and dephosphorylated conformations, alternately opening ion-binding sites externally (E2) or internally (E1). With subsaturating [Na+]oand [K+]o, the phosphorylated E2P conformation passively imports protons generating an inward current (IH), which may be exacerbated in NKA-subunit mutations associated with human disease. To elucidate the mechanisms of IH, we studied the effects of intracellular ligands (transported ions, nucleotides, and beryllium fluoride) on IHand, for comparison, on transient currents measured at normal Na+o(QNa). Utilizing inside-out patches from Xenopus oocytes heterologously expressing NKA, we observed that 1) in the presence of Na+i, IHand QNawere both activated by ATP, but not ADP; 2) the [Na+]idependence of IHin saturating ATP showed K0.5,Na= 1.8 ± 0.2 mM and the [ATP] dependence at saturating [Na+]iyielded K0.5,ATP= 48 ± 11 μM (in comparison, Na+i-dependent QNayields K0.5,Na= 0.8 ± 0.2 mM and K0.5,ATP= 0.43 ± 0.03 μM; 3) ATP activated IHin the presence of K+i(∼15% of the IHobserved in Na+i) only when Mg2+iwas also present; and 4) beryllium fluoride induced maximal IHeven in the absence of nucleotide. These data indicate that IHoccurs when NKA is in an externally open E2P state with nucleotide bound, a conformation that can be reached through forward Na/K pump phosphorylation of E1, with Na+iand ATP, or by backward binding of K+ito E1, which drives the pump to the occluded E2(2K), where free Pi(at the micromolar levels found in millimolar ATP solutions) promotes external release of occluded K+by backdoor NKA phosphorylation. Maximal IHthrough beryllium-fluorinated NKA indicates that this complex mimics ATP-bound E2P states.
authors.affiliation: Department of Biological Sciences, Illinois State University, Normal, Illinois; Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas.
authors.forename: Kevin S
authors.initials: KS
authors.lastname: Stanley
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas.
authors.forename: Dylan J
authors.initials: DJ
authors.lastname: Meyer
authors.affiliation: Department of Biological Sciences, Illinois State University, Normal, Illinois.
authors.forename: Craig
authors.initials: C
authors.lastname: Gatto
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas. Electronic address: pablo.artigas@ttuhsc.edu.
authors.forename: Pablo
authors.initials: P
authors.lastname: Artigas
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001608:Beryllium / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D055724:Electrophysiological Phenomena / Q000187:drug effects; D005459:Fluorides / Q000494:pharmacology; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism*; D011522:Protons; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014981:Xenopus
chemical_list: D011522:Protons; C035982:beryllium fluoride; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D001608:Beryllium; D005459:Fluorides