Abstract:The focus of this article is on progress in establishing structure-function relationships through site-directed mutagenesis and direct binding assay of Tl(+), Rb(+), K(+), Na(+), Mg(2+) or free ATP at equilibrium in Na,K-ATPase. Direct binding may identify residues coordinating cations in the E(2)[2K] or E(1)P[3Na] forms of the ping-pong reaction sequence and allow estimates of their contributions to the change of Gibbs free energy of binding. This is required to understand the molecular basis for the pronounc… Show more
“…The true effect on Na ϩ binding to E 1 may actually be more pronounced than revealed by the 3-fold decrease in apparent affinity, because it is masked by displacement of the conformational equilibrium in favor of E 1 . The 3-fold reduction or more of Na ϩ affinity is of the same magnitude as previously detected for some of the mutants with alterations to residues proposed to contribute to coordination of Na ϩ in the cation binding pocket (11,15,20).…”
supporting
confidence: 76%
“…Previous mutational studies of P-type ATPases have pinpointed highly conserved residues with oxygen-containing side chains in transmembrane segments M4, M5, and M6 as essential to cation binding and occlusion (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). The high-resolution crystal structure of the Ca 2ϩ -ATPase with bound Ca 2ϩ revealed that indeed these residues donate Ca 2ϩ ligands in the binding pocket (2).…”
Glu282 located in the NH 2 -terminal part of transmembrane helix M3 of the Na ؉ ,K ؉ -ATPase was replaced by alanine, glycine, leucine, lysine, aspartate, or glutamine, and the effects of the mutations on the overall and partial reactions of the enzyme were analyzed. The mutations affected at least 3 important functions of the Na ؉ ,K ؉ -ATPase: (i) the conformational transitions between E 1 and E 2 forms of dephospho-and phosphoenzyme, (ii) Na ؉ binding at the cytoplasmically facing sites of E 1 , and (iii) long-range interaction controlling dephosphorylation. In mutants Glu 282 3 Lys and Glu 282 3 Asp, the E 1 form was favored during ATP hydrolysis, whereas the E 2 form was favored in Glu 282 3 Ala and Glu 282 3 Gly. Regardless of the change of conformational equilibrium, all the mutants displayed a reduced apparent affinity for Na ؉ , at least 3-fold for Glu 282 3 Lys and Glu 282 3 Asp, suggesting a direct effect on the Na ؉ binding properties of E 1 . Glu 282 3 Ala and Glu 282 3 Gly exhibited an extraordinary high rate of ATP hydrolysis in the mere presence of Na ؉ without K ؉ ("Na ؉ -ATPase activity"), because of an increased rate of dephosphorylation of E 2 P. These results are in accordance with the hypothesis that Glu 282 is involved in the communication between the cation binding pocket and the catalytic site and in control of the cytoplasmic entry pathway for Na ؉ .
“…The true effect on Na ϩ binding to E 1 may actually be more pronounced than revealed by the 3-fold decrease in apparent affinity, because it is masked by displacement of the conformational equilibrium in favor of E 1 . The 3-fold reduction or more of Na ϩ affinity is of the same magnitude as previously detected for some of the mutants with alterations to residues proposed to contribute to coordination of Na ϩ in the cation binding pocket (11,15,20).…”
supporting
confidence: 76%
“…Previous mutational studies of P-type ATPases have pinpointed highly conserved residues with oxygen-containing side chains in transmembrane segments M4, M5, and M6 as essential to cation binding and occlusion (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). The high-resolution crystal structure of the Ca 2ϩ -ATPase with bound Ca 2ϩ revealed that indeed these residues donate Ca 2ϩ ligands in the binding pocket (2).…”
Glu282 located in the NH 2 -terminal part of transmembrane helix M3 of the Na ؉ ,K ؉ -ATPase was replaced by alanine, glycine, leucine, lysine, aspartate, or glutamine, and the effects of the mutations on the overall and partial reactions of the enzyme were analyzed. The mutations affected at least 3 important functions of the Na ؉ ,K ؉ -ATPase: (i) the conformational transitions between E 1 and E 2 forms of dephospho-and phosphoenzyme, (ii) Na ؉ binding at the cytoplasmically facing sites of E 1 , and (iii) long-range interaction controlling dephosphorylation. In mutants Glu 282 3 Lys and Glu 282 3 Asp, the E 1 form was favored during ATP hydrolysis, whereas the E 2 form was favored in Glu 282 3 Ala and Glu 282 3 Gly. Regardless of the change of conformational equilibrium, all the mutants displayed a reduced apparent affinity for Na ؉ , at least 3-fold for Glu 282 3 Lys and Glu 282 3 Asp, suggesting a direct effect on the Na ؉ binding properties of E 1 . Glu 282 3 Ala and Glu 282 3 Gly exhibited an extraordinary high rate of ATP hydrolysis in the mere presence of Na ؉ without K ؉ ("Na ؉ -ATPase activity"), because of an increased rate of dephosphorylation of E 2 P. These results are in accordance with the hypothesis that Glu 282 is involved in the communication between the cation binding pocket and the catalytic site and in control of the cytoplasmic entry pathway for Na ؉ .
“…DISCUSSION We have taken advantage of the functional differences and the high degree of sequence homology between the H,K-and Na,K-ATPases to attempt to identify the determinant of the electrogenicity of cation transport by the group IIc P-ATPases. In the fifth transmembrane segment, several amino acid residues have been shown to play a role in cation binding in both the H,K-and Na,K-ATPases (22,23,(25)(26)(27)(28)(29)(30)(31) and also in SERCA (32). The middle of the fifth transmembrane segment region is highly similar between the H,K-and Na,K-ATPases (Fig.…”
Section: Electrogenic Transport By the Lys 800 Mutants Of The Hkatpamentioning
confidence: 90%
“…Only the S782A mutant had a very small but significant 86 Rb uptake of 2.6 Ϯ 1.1 pmol/min, whereas the wild-type Na,KATPase expressed a transport activity of 45.1 Ϯ 8.6 pmol/min, similar to that of the wild-type H,K-ATPase. A low affinity for extracellular K ϩ has been observed with mutants of the corresponding position (Ser 775 ) in ␣ 1 Na,K-ATPase from other species (21)(22)(23). Assuming a similar effect of homologous mutations in the Bufo ␣ 1 Na,K-pump, we studied the transport function of the Ser 782 mutants at a higher (40 mM) concentration of K ϩ .…”
Section: Expression Of the B Marinus Nak-and Hk-atpase Mutants-mentioning
“…During ATP hydrolysis, Ptype ATPases are phosphorylated at a highly conserved aspartate residue (Axelsen and Palmgren, 1998). Na þ /K þ ATPase is composed of two mandatory subunits, the a and b subunits (Jorgensen and Pedersen, 2001). The a subunit contains 10 transmembrane segments, including Na þ /K þ ion binding domains, cytoplasmic phosphorylation sites, inhibitor (ouabain and digitoxin) and activator binding domain (Kaplan, 2002).…”
Very recently, voltage-clamp fluorometry (VCF) protocols have been developed to observe the membrane proteins responsible for carrying the ventricular ionic currents that form the action potential (AP), including those carried by the cardiac Na þ channel, Na V 1.5, the L-type Ca 2þ channel, Ca V 1.2, the Na þ /K þ ATPase, and the rapid and slow components of the delayed rectifier, K V 11.1 and K V 7.1. This development is significant, because VCF enables simultaneous observation of ionic current kinetics with conformational changes occurring within specific channel domains. The ability gained from VCF, to connect nanoscale molecular movement to ion channel function has revealed how the voltage-sensing domains (VSDs) control ion flux through channel pores, mechanisms of post-translational regulation and the molecular pathology of inherited mutations. In the future, we expect that this data will be of great use for the creation of multi-scale computational AP models that explicitly represent ion channel conformations, connecting molecular, cell and tissue electrophysiology. Here, we review the VCF protocol, recent results, and discuss potential future developments, including potential use of these experimental findings to create novel computational models.
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