TY - JOUR

T1 - Structural modeling and electron paramagnetic resonance spectroscopy of the human Na+/H+ exchanger isoform 1, NHE1

AU - Nygaard, Eva B

AU - Lagerstedt, Jens O

AU - Bjerre, Gabriel

AU - Shi, Biao

AU - Budamagunta, Madhu

AU - Poulsen, Kristian A

AU - Lundby, Stine Meinild

AU - Rigor, Robert R

AU - Voss, John C

AU - Cala, Peter M

AU - Pedersen, Stine Helene Falsig

PY - 2011

Y1 - 2011

N2 - We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na(+)/H(+) exchanger, hNHE1 (Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., and Cala, P. M. (2007) J. Biol. Chem. 282, 19716-19727). Here, we present a structural model of the transmembrane part of hNHE1 that further supports this conclusion. The hNHE1 model was based on the crystal structure of the Escherichia coli Na(+)/H(+) antiporter, NhaA, and previous cysteine scanning accessibility studies of hNHE1 and was validated by EPR spectroscopy of spin labels in TM IV and TM XI, as well as by functional analysis of hNHE1 mutants. Removal of all endogenous cysteines in hNHE1, introduction of the mutations A173C (TM IV) and/or I461C (TM XI), and expression of the constructs in mammalian cells resulted in functional hNHE1 proteins. The distance between these spin labels was ~15 A, confirming that TM IV and TM XI are in close proximity. This distance was decreased both at pH 5.1 and in the presence of the NHE1 inhibitor cariporide. A similar TM IV·TM XI distance and a similar change upon a pH shift were found for the cariporide-insensitive Pleuronectes americanus (pa) NHE1; however, in paNHE1, cariporide had no effect on TM IV·TM XI distance. The central role of the TM IV·TM XI arrangement was confirmed by the partial loss of function upon mutation of Arg(425), which the model predicts stabilizes this arrangement. The data are consistent with a role for TM IV and TM XI rearrangements coincident with ion translocation and inhibitor binding by hNHE1.

AB - We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na(+)/H(+) exchanger, hNHE1 (Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., and Cala, P. M. (2007) J. Biol. Chem. 282, 19716-19727). Here, we present a structural model of the transmembrane part of hNHE1 that further supports this conclusion. The hNHE1 model was based on the crystal structure of the Escherichia coli Na(+)/H(+) antiporter, NhaA, and previous cysteine scanning accessibility studies of hNHE1 and was validated by EPR spectroscopy of spin labels in TM IV and TM XI, as well as by functional analysis of hNHE1 mutants. Removal of all endogenous cysteines in hNHE1, introduction of the mutations A173C (TM IV) and/or I461C (TM XI), and expression of the constructs in mammalian cells resulted in functional hNHE1 proteins. The distance between these spin labels was ~15 A, confirming that TM IV and TM XI are in close proximity. This distance was decreased both at pH 5.1 and in the presence of the NHE1 inhibitor cariporide. A similar TM IV·TM XI distance and a similar change upon a pH shift were found for the cariporide-insensitive Pleuronectes americanus (pa) NHE1; however, in paNHE1, cariporide had no effect on TM IV·TM XI distance. The central role of the TM IV·TM XI arrangement was confirmed by the partial loss of function upon mutation of Arg(425), which the model predicts stabilizes this arrangement. The data are consistent with a role for TM IV and TM XI rearrangements coincident with ion translocation and inhibitor binding by hNHE1.

KW - Amino Acid Sequence

KW - Animals

KW - Arginine

KW - Cation Transport Proteins

KW - Cell Line

KW - Cell Membrane

KW - Cricetinae

KW - Cricetulus

KW - Electron Spin Resonance Spectroscopy

KW - Flounder

KW - Humans

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Mutation

KW - Protein Structure, Tertiary

KW - Sodium-Hydrogen Antiporter

U2 - 10.1074/jbc.M110.159202

DO - 10.1074/jbc.M110.159202

M3 - Journal article

C2 - 20974853

VL - 286

SP - 634

EP - 648

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 1

ER -