Alanine Insertion Scanning Mutagenesis of Lactose Permease Transmembrane Helices

Braun, Paula; Persson, Bengt L.; Kaback, H. Ronald; Heijne, Gunnar von

doi:10.1074/jbc.272.47.29566

Cited by 36 publications

(42 citation statements)

References 37 publications

(40 reference statements)

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“…1A). Alanine insertion into an ␣ helix is believed to dislocate the N-terminal part of the helix by 100°with respect to the C-terminal part and consequently impede the structural and/or functional continuity of the helix (16). Tryptophan scanning has been used successfully to determine functionally important amino acids in integral membrane proteins (16 -18).…”

Section: Expression and Processing Of Fxyd7 Tm Mutants-mentioning

confidence: 99%

Role of the Transmembrane Domain of FXYD7 in Structural and Functional Interactions with Na,K-ATPase

Crambert

Thuillard

et al. 2005

Journal of Biological Chemistry

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Members of the FXYD family are tissue-specific regulators of the Na,K-ATPase. Here, we have investigated the contribution of amino acids in the transmembrane (TM) domain of FXYD7 to the interaction with Na,K-ATPase. Twenty amino acids of the TM domain were replaced individually by tryptophan, and combined mutations and alanine insertion mutants were constructed. Wild type and mutant FXYD7 were expressed in Xenopus oocytes with Na,KATPase. Mutational effects on the stable association with Na,KATPase and on the functional regulation of Na,K-ATPase were determined by co-immunoprecipitation and two-electrode voltage clamp techniques, respectively. Most residues important for the structural and functional interaction of FXYD7 are clustered in a face of the TM helix containing the two conserved glycine residues, but others are scattered over two-thirds of the FXYD TM helix. By using the free energy from the hydrolysis of one ATP molecule, Na,K-ATPase, a ubiquitous P-type ATPase, transports three Na ϩ out of the cell in exchange for two K ϩ into the cell. The minimal functional unit of Na,K-ATPase consists of a catalytic ␣ subunit and a regulatory ␤ subunit. Four ␣ and 3 ␤ isoforms exist and are expressed in a tissuespecific manner.The major physiological role of Na,K-ATPase is to create and maintain the Na ϩ /K ϩ gradient across the cell membrane, which is important for the maintenance of cell volume and membrane potential. Moreover, the Na ϩ gradient serves as an energy source for numerous secondary transport systems. Finally, Na,K-ATPase is essential for specialized functions of various tissues. In renal epithelial cells, Na,K-ATPase is exclusively expressed at the basolateral membrane and becomes the driving force for transepithelial Na ϩ reabsorption. In nervous tissue, Na,K-ATPase restores the Na ϩ and K ϩ gradients during action potentials and thus ensures neuronal excitability. In skeletal and heart muscle, the Na ϩ gradient coupled to Na ϩ /Ca 2ϩ exchanger activity controls the intracellular Ca 2ϩ concentration, which influences muscle contractility. The activity of Na,K-ATPase is subjected to short-and long-term regulation (1) mediated by the intracellular Na ϩ concentration, neurotransmitters, and hormones. Recently, members of the FXYD family (2) have been shown to be tissue-specific regulators of the Na,K-ATPase (3). FXYD proteins are small membrane proteins that are characterized by an N-terminal FXYD motif, two highly conserved glycine residues in the TM domain, and a serine residue at the cytoplasmic end of the TM domain. FXYD7, an O-glycosylated protein, is only expressed in brain, in both neurons and glial cells (4). FXYD7 associates with ␣␤1 isozymes, but not with ␣␤2 isozymes, when expressed in Xenopus oocytes. Stable association of FXYD7 with Na,K-ATPase modifies its apparent affinity for external K ϩ by increasing by ϳ2-fold the K1 ⁄ 2 value for K ϩ over a large range of membrane potentials in the presence and absence of external Na ϩ . In addition, FXYD7 modifies the apparent affinity for extrace...

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Section: Expression and Processing Of Fxyd7 Tm Mutants-mentioning

confidence: 99%

Role of the Transmembrane Domain of FXYD7 in Structural and Functional Interactions with Na,K-ATPase

Crambert

Thuillard

et al. 2005

Journal of Biological Chemistry

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“…It has been observed that inserting single amino acids near the central core of transmembrane helices in lactose permease disrupts protein function (30). Three mutants were constructed with an alanine inserted in different hydrophobic sections between loop f and Ser-788.…”

Section: Topology Of the Namentioning

confidence: 99%

A New Topological Model of the Cardiac Sarcolemmal Na+-Ca2+ Exchanger

Nicoll

Ottolia

Lü

et al. 1999

Journal of Biological Chemistry

188

162

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The current topological model of the Na ؉ -Ca 2؉ exchanger consists of 11 transmembrane segments with extracellular loops a, c, e, g, i, and k and cytoplasmic loops b, d, f, h, and j. Cytoplasmic loop f, which plays a role in regulating the exchanger, is large and separates the first five from the last six transmembrane segments. We have tested this topological model by mutating residues near putative transmembrane segments to cysteine and then examining the effects of intracellular and extracellular applications of sulfhydryl-modifying reagents on exchanger activity. To aid in our topological studies, we also constructed a cysteineless Na ؉ -Ca 2؉ exchanger. This mutant is fully functional in Na ؉ gradientdependent 45 Ca 2؉ uptake measurements and displays wild-type regulatory properties. It is concluded that the 15 endogenous cysteine residues are not essential for either activity or regulation of the exchanger. Our data support the current model by placing loops c and e at the extracellular surface and loops d, j, and l at the intracellular surface. However, the data also support placing Ser-788 of loop h at the extracellular surface and Gly-837 of loop i at the intracellular surface. To account for these data, we propose a revision of the model that places transmembrane segment 6 in cytoplasmic loop f. Additionally, we propose that putative transmembrane segment 9 does not span the membrane, but may form a "P-loop"-like structure.

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“…However, in dealing with disease mutants, multiple amino acid properties are changed, possibly altering function by way of what was introduced, rather than what was taken out. Systematic mutation to a relatively neutral amino acid like alanine (Braun et al 1997) or cysteine, as done in the SCAM studies, is a more interpretable approach, particularly if the overall distribution of sensitive sites on the helices are considered as an indicator of transmembrane domain interactions. Thus, the distribution of cysteine substitutions in the SCAM analysis of Skerrett et al (2002) and Toloue (2007) that led to nonfunctional gap junctions or channels with aberrant properties may provide the strongest indication yet as to the likely sites of interhelical interaction within the connexin.…”

Section: Introductionmentioning

confidence: 99%

Site-Directed Mutagenesis Reveals Putative Regions of Protein Interaction within the Transmembrane Domains of Connexins

Toloue

Woolwine

Karcz

et al. 2008

Cell Communication & Adhesion

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Through cysteine-scanning mutagenesis, the authors have compared sites within the transmembrane domains of two connexins, one from the α-class (Cx50) and one from the β-class (Cx32), where amino acid substitution disrupts the function of gap junction channels. In Cx32, 11 sites resulted in no channel function, or an aberrant voltage gating phenotype referred to as "reverse gating," whereas in Cx50, 7 such sites were identified. In both connexins, the sites lie along specific faces of transmembrane helices, suggesting that these may be sites of transmembrane domain interactions. In Cx32, one broad face of the M1 transmembrane domain and a narrower, polar face of M3 were identified, including one site that was shown to come into close apposition with M4 in the closed state. In Cx50, the same face of M3 was identified, but sensitive sites in M1 differed from Cx32. Many fewer sites in M1 disrupted channel function in Cx50, and those that did were on a different helical face to the sensitive sites in Cx32. A more in depth study of two sites in M1 and M2 of Cx32 showed that side-chain length or branching are important for maintenance of normal channel behavior, consistent with this being a site of transmembrane domain interaction.

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Alanine Insertion Scanning Mutagenesis of Lactose Permease Transmembrane Helices

Cited by 36 publications

References 37 publications

Role of the Transmembrane Domain of FXYD7 in Structural and Functional Interactions with Na,K-ATPase

Role of the Transmembrane Domain of FXYD7 in Structural and Functional Interactions with Na,K-ATPase

A New Topological Model of the Cardiac Sarcolemmal Na+-Ca2+ Exchanger

Site-Directed Mutagenesis Reveals Putative Regions of Protein Interaction within the Transmembrane Domains of Connexins

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