HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 11, 10210-10218, March 18, 2005

This Article Full Text Full Text (PDF) All Versions of this Article: 280/11/10210    most recent M411214200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Toustrup-Jensen, M. Articles by Vilsen, B. Search for Related Content PubMed PubMed Citation Articles by Toustrup-Jensen, M. Articles by Vilsen, B. Social Bookmarking                      What's this?

Interaction between the Catalytic Site and the A-M3 Linker Stabilizes E₂/E₂P Conformational States of Na⁺,K⁺-ATPase^*

Mads Toustrup-Jensen and Bente Vilsen

From the Department of Physiology, Institute of Physiology and Biophysics, University of Aarhus, Ole Worms Allé 160, DK-8000 Aarhus C, Denmark

The consequences of mutations Ile²⁶⁵ Ala, Thr²⁶⁷ Ala, Gly²⁷¹ Ala, and Gly²⁷⁴ Ala for the partial reaction steps of the Na⁺,K⁺-ATPase transport cycle were analyzed. The mutated residues are part of the long loop ("A-M3 linker") connecting the cytoplasmic A-domain with transmembrane segment M3. It was found that mutation Ile²⁶⁵ Ala displaces the E₁-E₂ and E₁P-E₂P equilibria in favor of E₁/E₁P, whereas mutations Thr²⁶⁷ Ala, Gly²⁷¹ Ala, and Gly²⁷⁴ Ala displace these conformational equilibria in favor of E₂/E₂P. The mutations affect both the rearrangement of the cytoplasmic domains (seen by changes in phosphoenzyme properties and apparent ATP/vanadate affinities) and the membrane sector (indicated by change in K⁺/Rb⁺ deocclusion rate). Destabilization of E₂/E₂P in Ile²⁶⁵ Ala, as well as a direct effect on the intrinsic affinity of the E₂ form for vanadate, may be explained on the basis of the E₂ crystal structures of the Ca²⁺-ATPase, showing interaction of the equivalent isoleucine with conserved residues near the catalytic region of the P-domain. The rate of phosphorylation from ATP was unaffected in Ile²⁶⁵ Ala, indicating a lack of interference with the catalytic function in E₁/E₁P. The effects of mutations Thr²⁶⁷ Ala, Gly²⁷¹ Ala, and Gly²⁷⁴ Ala provide the first evidence in the literature of a relative stabilization of E₂/E₂P resulting from perturbation of the A-M3 linker region. These mutations may lead to increased strain of the A-M3 linker in E₁/E₁P, increased stability of the A3 helix of the A-M3 linker in E₂/E₂P, and/or a change of the orientation of the A3 helix, facilitating its interaction with the P-domain.

Received for publication, September 30, 2004 , and in revised form, November 26, 2004.

^* This work was supported by grants from the Danish Medical Research Council, the Novo Nordisk Foundation, Denmark, the Lundbeck Foundation, Denmark, and the Research Foundation of Aarhus University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 45-89-42-28-32; Fax: 45-86-12-90-65; E-mail: bv{at}fi.au.dk.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				V. R. Schack, J. P. Morth, M. S. Toustrup-Jensen, A. N. Anthonisen, P. Nissen, J. P. Andersen, and B. Vilsen Identification and Function of a Cytoplasmic K+ Site of the Na+, K+-ATPase J. Biol. Chem., October 10, 2008; 283(41): 27982 - 27990. [Abstract] [Full Text] [PDF]

				G. Wang, K. Yamasaki, T. Daiho, and H. Suzuki Critical Hydrophobic Interactions between Phosphorylation and Actuator Domains of Ca2+-ATPase for Hydrolysis of Phosphorylated Intermediate J. Biol. Chem., July 15, 2005; 280(28): 26508 - 26516. [Abstract] [Full Text] [PDF]