Structural and Functional Analysis of Transmembrane XI of the NHE1
Isoform of the Na+/H+
Exchanger |
| |
Authors: | Brian L Lee Xiuju Li Yongsheng Liu Brian D Sykes and Larry Fliegel |
| |
Institution: | Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada |
| |
Abstract: | The Na+/H+ exchanger isoform 1 is a ubiquitously
expressed integral membrane protein that regulates intracellular pH in mammals
by extruding an intracellular H+ in exchange for one extracellular
Na+. We characterized structural and functional aspects of the
critical transmembrane (TM) segment XI (residues 449-470) by using cysteine
scanning mutagenesis and high resolution NMR. Each residue of TM XI was
mutated to cysteine in the background of the cysteine-less protein and the
sensitivity to water-soluble sulfhydryl reactive compounds MTSET
((2-(trimethylammonium) ethyl)methanethiosulfonate) and MTSES
((2-sulfonatoethyl) methanethiosulfonate) was determined for those residues
with at least moderate activity remaining. Of the residues tested, only
proteins with mutations L457C, I461C, and L465C were inhibited by MTSET. The
activity of the L465C mutant was almost completely eliminated, whereas that of
the L457C and I461C mutants was partially affected. The structure of a peptide
representing TM XI (residues Lys447-Lys472) was
determined using high resolution NMR spectroscopy in dodecylphosphocholine
micelles. The structure consisted of helical regions between
Asp447-Tyr454 and Phe460-Lys471 at
the N and C termini of the peptide, respectively, connected by a region with
poorly defined, irregular structure consisting of residues
Gly455-Gly459. TM XI of NHE1 had a structural similarity
to TM XI of the Escherichia coli Na+/H+
exchanger NhaA. The results suggest that TM XI is a discontinuous helix, with
residue Leu465 contributing to the pore.The mammalian Na+/H+ exchanger isoform 1
(NHE1)4 is a
ubiquitous integral membrane protein that regulates intracellular pH. It
mediates removal of a single intracellular proton in exchange for an
extracellular sodium ion (1).
NHE1 has many functions aside from protection of cells from intracellular
acidification (2). It promotes
cell growth and differentiation
(3), regulates sodium fluxes
and cell volume after challenge by osmotic shrinkage
(4), and has been demonstrated
to be involved in modulating cell motility
(5). In addition its activity
is important in invasiveness of neoplastic breast cancer cells
(6). NHE1 also plays critical
roles in heart disease. It has a contributing role in heart hypertrophy and in
the damage that occurs during ischemia and reperfusion. Inhibition of NHE1
with Na+/H+ exchanger inhibitors protects the myocardium
during various disease states
(7-10).NHE1 is composed of two general regions, an N-terminal membrane domain of
∼500 amino acids and a C-terminal regulatory domain of ∼315 amino
acids (1,
8). The membrane domain is
responsible for ion movement and an analysis of topology by cysteine scanning
accessibility suggested it has 3 membrane-associated segments and 12 integral
transmembrane segments (11)
(). The
mechanism of transport of the membrane domain is of great interest both from a
scientific viewpoint and in the design of improved NHE1 inhibitors that may be
necessary for clinical use (1).
In this regard, we have recently characterized the functionally important
residues and the structure of both TM IV and TM VII. Prolines 167 and 168 of
TM IV were critical to NHE1 function
(12) and cysteine-scanning
mutagenesis was used to show that Phe161 is a pore lining residue
critical to transport. Analysis of the structure of TM IV showed that TM IV is
composed of one region of β-turns, an extended middle region including
Pro167-Pro168, and a helical region
(13). TM VII was much more
typical of a transmembrane helix although it was interrupted with a break in
the helix at the functionally critical residues
Gly261-Glu262
(14).Open in a separate windowModels of the Na+/H+ exchanger.
A, simplified topological model of the transmembrane domain of the
NHE1 isoform of the Na+/H+ exchanger as described
earlier (11). EL,
extracellular loop; IL, intracellular loop. B, model of amino acids
present in TM XI.Another important TM segment of the Na+/H+ exchanger
is TM XI ().
Several different lines of evidence have suggested that it is critical to NHE1
function. A recent study generated chimeras of NHE1 from various species and
found that a region including TM XI was important in determining NHE1
inhibitor sensitivity (15).
More specifically, mutagenesis of several amino acids of TM XI has shown that
it is likely involved in either ion transport or proper targeting to the
plasma membrane. Two mutants in TM XI, Y454C and R458C, are retained in the
endoplasmic reticulum (16). In
addition, mutation of Gly455 and Gly456 in TM XI shift
the pHi dependence of the exchanger to the alkaline side,
whereas mutation of Arg440 in intracellular loop 5 at the
N-terminal end of TM XI shifts the pHi dependence to make
it more acidic (17,
18). Also, the structure of
the bacterial Na+/H+ exchanger NhaA has been elucidated.
Both TM IV and TM XI play a critical role forming an assembly that cross, with
each being a helix, an extended polypeptide and a short helix
(19). We found that TM IV of
NHE1 has a similar structure and function to that of TM IV of NhaA
(2,
13), leaving open the
possibility that TM XI of NHE1 is also similar in structure and function to TM
XI of NhaA.For these reasons, we undertook a systematic examination of the structural
and functional aspects of TM XI of the NHE1 isoform of the
Na+/H+ exchanger. The sequence of human TM XI of NHE1 is
449QFIIAYGGLRGAIAFSLGYLLD470. In this study we use
cysteine scanning mutagenesis and site-specific mutagenesis to identify and
characterize critical pore lining residues of the protein. We also use nuclear
magnetic resonance (NMR) spectroscopy to characterize the structure of a
synthetic peptide representing TM XI in dodecylphosphocholine (DPC) micelles.
Evidence has suggested that TM segments of membrane proteins possess all the
structural information required to form their higher order structures in their
amino acid sequence (20). This
has been demonstrated in earlier studies on membrane protein segments such as
the cystic fibrosis transmembrane conductance regulator
(21), a fungal
G-protein-coupled receptor
(22), bacteriorhodopsin
(23,
24), and rhodopsin
(25), where it was shown that
isolated TM segments from membrane proteins had structures in good agreement
with the segments of the entire protein. Also, the use of DPC micelles has
been shown to be an excellent membrane mimetic environment for these studies
(26,
27). Our study identifies
Leu465 as contributing to the pore of the protein and shows that
the structure of TM XI consists of two helices corresponding to
Asp447-Tyr454 and Phe460-Lys471 at
the N and C termini, respectively, connected by a flexible region at residues
455-459. The structure of TM XI was similar to the x-ray structure of TM XI of
NhaA. |
| |
Keywords: | |
|
|