Structural and Functional Analysis of Transmembrane Segment VI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na⁺/H⁺ exchanger isoform 1 is a ubiquitously expressed integral membrane protein. It resides on the plasma membrane of cells and regulates intracellular pH in mammals by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. We characterized structural and functional aspects of the transmembrane segment (TM) VI (residues 227–249) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM VI was mutated to cysteine in the background of the cysteineless NHE1 protein, and the sensitivity to water-soluble sulfhydryl-reactive compounds (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and (2-sulfonatoethyl)methanethiosulfonate (MTSES) was determined for those residues with significant activity remaining. Three residues were essentially inactive when mutated to Cys: Asp²³⁸, Pro²³⁹, and Glu²⁴⁷. Of the remaining residues, proteins with the mutations N227C, I233C, and L243C were strongly inhibited by MTSET, whereas amino acids Phe²³⁰, Gly²³¹, Ala²³⁶, Val²³⁷, Ala²⁴⁴, Val²⁴⁵, and Glu²⁴⁸ were partially inhibited by MTSET. MTSES did not affect the activity of the mutant NHE1 proteins. The structure of a peptide representing TM VI was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. TM VI contains two helical regions oriented at an approximate right angle to each other (residues 229–236 and 239–250) surrounding a central unwound region. This structure bears a resemblance to TM IV of the Escherichia coli protein NhaA. The results demonstrate that TM VI of NHE1 is a discontinuous pore-lining helix with residues Asn²²⁷, Ile²³³, and Leu²⁴³ lining the translocation pore.     

Structure of human Na+/H+ exchanger NHE1 regulatory region in complex with calmodulin and Ca2+

The ubiquitous mammalian Na⁺/H⁺ exchanger NHE1 has critical functions in regulating intracellular pH, salt concentration, and cellular volume. The regulatory C-terminal domain of NHE1 is linked to the ion-translocating N-terminal membrane domain and acts as a scaffold for signaling complexes. A major interaction partner is calmodulin (CaM), which binds to two neighboring regions of NHE1 in a strongly Ca²⁺-dependent manner. Upon CaM binding, NHE1 is activated by a shift in sensitivity toward alkaline intracellular pH. Here we report the 2.23 Å crystal structure of the NHE1 CaM binding region (NHE1_CaMBR) in complex with CaM and Ca²⁺. The C- and N-lobes of CaM bind the first and second helix of NHE1_CaMBR, respectively. Both the NHE1 helices and the Ca²⁺-bound CaM are elongated, as confirmed by small angle x-ray scattering analysis. Our x-ray structure sheds new light on the molecular mechanisms of the phosphorylation-dependent regulation of NHE1 and enables us to propose a model of how Ca²⁺ regulates NHE1 activity.     

Chloride channel (Clc)-5 is necessary for exocytic trafficking of Na+/H+ exchanger 3 (NHE3)

ClC-5, a chloride/proton exchanger, is predominantly expressed and localized in subapical endosomes of the renal proximal tubule. Mutations of the CLCN5 gene cause Dent disease. The symptoms of Dent disease are replicated in Clcn5 knock-out mice. Absence of ClC-5 in mice is associated with reduced surface expression of NHE3 in proximal tubules. The molecular basis for this change is not fully understood. In this study, we investigated the mechanisms by which ClC-5 regulates trafficking of NHE3. Whether ClC-5-dependent endocytosis, exocytosis, or both contributed to the altered distribution of NHE3 was examined. First, NHE3 activity in proximal tubules of wild type (WT) and Clcn5 KO mice was determined by two-photon microscopy. Basal and dexamethasone-stimulated NHE3 activity of Clcn5 KO mice was decreased compared with that seen in WT mice, whereas the degree of inhibition of NHE3 activity by increasing cellular concentration of cAMP (forskolin) or Ca(2+) (A23187) was not different in WT and Clcn5 KO mice. Second, NHE3-dependent absorption of HCO(3)(-), measured by single tubule perfusion, was reduced in proximal tubules of Clcn5 KO mice. Third, by cell surface biotinylation, trafficking of NHE3 was examined in short hairpin RNA (shRNA) plasmid-transfected opossum kidney cells. Surface NHE3 was reduced in opossum kidney cells with reduced expression of ClC-5, whereas the total protein level of NHE3 did not change. Parathyroid hormone decreased NHE3 surface expression, but the extent of decrease and the rate of endocytosis observed in both scrambled and ClC-5 knockdown cells were not significantly different. However, the rates of basal and dexamethasone-stimulated exocytosis of NHE3 were attenuated in ClC-5 knockdown cells. These results show that ClC-5 plays an essential role in exocytosis of NHE3.     

Phosphoinositide binding differentially regulates NHE1 Na+/H+ exchanger-dependent proximal tubule cell survival

Tubular atrophy predicts chronic kidney disease progression, and is caused by proximal tubular epithelial cellcaused by proximal tubular epithelial cell (PTC) apoptosis. The normally quiescent Na(+)/H(+) exchanger-1 (NHE1) defends against PTC apoptosis, and is regulated by PI(4,5)P(2) binding. Because of the vast array of plasma membrane lipids, we hypothesized that NHE1-mediated cell survival is dynamically regulated by multiple anionic inner leaflet phospholipids. In membrane overlay and surface plasmon resonance assays, the NHE1 C terminus bound phospholipids with low affinity and according to valence (PIP(3) > PIP(2) > PIP = PA > PS). NHE1-phosphoinositide binding was enhanced by acidic pH, and abolished by NHE1 Arg/Lys to Ala mutations within two juxtamembrane domains, consistent with electrostatic interactions. PI(4,5)P(2)-incorporated vesicles were distributed to apical and lateral PTC domains, increased NHE1-regulated Na(+)/H(+) exchange, and blunted apoptosis, whereas NHE1 activity was decreased in cells enriched with PI(3,4,5)P(3), which localized to basolateral membranes. Divergent PI(4,5)P(2) and PI(3,4,5)P(3) effects on NHE1-dependent Na(+)/H(+) exchange and apoptosis were confirmed by selective phosphoinositide sequestration with pleckstrin homology domain-containing phospholipase Cδ and Akt peptides, PI 3-kinase, and Akt inhibition in wild-type and NHE1-null PTCs. The results reveal an on-off switch model, whereby NHE1 toggles between weak interactions with PI(4,5)P(2) and PI(3,4,5)P(3). In response to apoptotic stress, NHE1 is stimulated by PI(4,5)P(2), which leads to PI 3-kinase activation, and PI(4,5)P(2) phosphorylation. The resulting PI(3,4,5)P(3) dually stimulates sustained, downstream Akt survival signaling, and dampens NHE1 activity through competitive inhibition and depletion of PI(4,5)P(2).     

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

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.     

CK2 phosphorylation of an acidic Ser/Thr di-isoleucine motif in the Na+/H+ exchanger NHE5 isoform promotes association with beta-arrestin2 and endocytosis

Internalization of the Na(+)/H(+) exchanger NHE5 into recycling endosomes is enhanced by the endocytic adaptor proteins β-arrestin1 and -2, best known for their preferential recognition of ligand-activated G protein-coupled receptors (GPCRs). However, the mechanism underlying their atypical association with non-GPCRs, such as NHE5, is unknown. In this study, we identified a highly acidic, serine/threonine-rich, di-isoleucine motif (amino acids 697-723) in the cytoplasmic C terminus of NHE5 that is recognized by β-arrestin2. Gross deletions of this site decreased the state of phosphorylation of NHE5 as well as its binding and responsiveness to β-arrestin2 in intact cells. More refined in vitro analyses showed that this site was robustly phosphorylated by the acidotropic protein kinase CK2, whereas other kinases, such as CK1 or the GPCR kinase GRK2, were considerably less potent. Simultaneous mutation of five Ser/Thr residues within 702-714 to Ala ((702)ST/AA(714)) abolished phosphorylation and binding of β-arrestin2. In transfected cells, the CK2 catalytic α subunit formed a complex with NHE5 and decreased wild-type but not (702)ST/AA(714) NHE5 activity, further supporting a regulatory role for this kinase. The rate of internalization of (702)ST/AA(714) was also diminished and relatively insensitive to overexpression of β-arrestin2. However, unlike in vitro, this mutant retained its ability to form a complex with β-arrestin2 despite its lack of responsiveness. Additional mutations of two di-isoleucine-based motifs (I697A/L698A and I722A/I723A) that immediately flank the acidic cluster, either separately or together, were required to disrupt their association. These data demonstrate that discrete elements of an elaborate sorting signal in NHE5 contribute to β-arrestin2 binding and trafficking along the recycling endosomal pathway.     

Novel Phorbol Ester-binding Motif Mediates Hormonal Activation of Na+/H+ Exchanger

Protein kinase C (PKC) is considered crucial for hormonal Na⁺/H⁺ exchanger (NHE1) activation because phorbol esters (PEs) strongly activate NHE1. However, here we report that rather than PKC, direct binding of PEs/diacylglycerol to the NHE1 lipid-interacting domain (LID) and the subsequent tighter association of LID with the plasma membrane mainly underlies NHE1 activation. We show that (i) PEs directly interact with the LID of NHE1 in vitro, (ii) like PKC, green fluorescent protein (GFP)-labeled LID translocates to the plasma membrane in response to PEs and receptor agonists, (iii) LID mutations markedly inhibit these interactions and PE/receptor agonist-induced NHE1 activation, and (iv) PKC inhibitors ineffectively block NHE1 activation, except staurosporin, which itself inhibits NHE1 via LID. Thus, we propose a PKC-independent mechanism of NHE1 regulation via a PE-binding motif previously unrecognized.     

Keeping It Simple,Transport Mechanism and pH Regulation in Na+/H+ Exchangers

Na⁺/H⁺ exchangers are essential for regulation of intracellular proton and sodium concentrations in all living organisms. We examined and experimentally verified a kinetic model for Na⁺/H⁺ exchangers, where a single binding site is alternatively occupied by Na⁺ or one or two H⁺ ions. The proposed transport mechanism inherently down-regulates Na⁺/H⁺ exchangers at extreme pH, preventing excessive cytoplasmic acidification or alkalinization. As an experimental test system we present the first electrophysiological investigation of an electroneutral Na⁺/H⁺ exchanger, NhaP1 from Methanocaldococcus jannaschii (MjNhaP1), a close homologue of the medically important eukaryotic NHE Na⁺/H⁺ exchangers. The kinetic model describes the experimentally observed substrate dependences of MjNhaP1, and the transport mechanism explains alkaline down-regulation of MjNhaP1. Because this model also accounts for acidic down-regulation of the electrogenic NhaA Na⁺/H⁺ exchanger from Escherichia coli (EcNhaA, shown in a previous publication) we conclude that it applies generally to all Na⁺/H⁺ exchangers, electrogenic as well as electroneutral, and elegantly explains their pH regulation. Furthermore, the electrophysiological analysis allows insight into the electrostatic structure of the translocation complex in electroneutral and electrogenic Na⁺/H⁺ exchangers.     

Calmodulin kinase II constitutively binds, phosphorylates, and inhibits brush border Na+/H+ exchanger 3 (NHE3) by a NHERF2 protein-dependent process

The epithelial brush border (BB) Na(+)/H(+) exchanger 3 (NHE3) accounts for most renal and intestinal Na(+) absorption. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibits NHE3 activity under basal conditions in intact intestine, acting in the BB, but the mechanism is unclear. We now demonstrate that in both PS120 fibroblasts and polarized Caco-2BBe cells expressing NHE3, CaMKII inhibits basal NHE3 activity, because the CaMKII-specific inhibitors KN-93 and KN-62 stimulate NHE3 activity. This inhibition requires NHERF2. CaMKIIγ associates with NHE3 between aa 586 and 605 in the NHE3 C terminus in a Ca(2+)-dependent manner, with less association when Ca(2+) is increased. CaMKII inhibits NHE3 by an effect on its turnover number, not changing surface expression. Back phosphorylation demonstrated that NHE3 is phosphorylated by CaMKII under basal conditions. This overall phosphorylation of NHE3 is not affected by the presence of NHERF2. Amino acids downstream of NHE3 aa 690 are required for CaMKII to inhibit basal NHE3 activity, and mutations of the three putative CaMKII phosphorylation sites downstream of aa 690 each prevented KN-93 stimulation of NHE3 activity. These studies demonstrate that CaMKIIγ is a novel NHE3-binding protein, and this association is reduced by elevated Ca(2+). CaMKII inhibits basal NHE3 activity associated with phosphorylation of NHE3 by effects requiring aa downstream of NHE3 aa 690 and of the CaMKII-binding site on NHE3. CaMKII binding to and phosphorylation of the NHE3 C terminus are parts of the physiologic regulation of NHE3 that occurs in fibroblasts as well as in the BB of an intestinal Na(+)-absorptive cell.     

Structure and function of the NHE1 isoform of the Na+/H+ exchanger.

The Na+/H+ exchanger is a ubiquitous, integral membrane protein involved in pH regulation. It removes intracellular acid, exchanging a proton for an extracellular sodium ion. There are seven known isoforms of this protein that are the products of distinct genes. The first isoform discovered (NHE1) is ubiquitously distributed throughout the plasma membrane of virtually all tissues. It plays many different physiological roles in mammals, including important functions in regulation of intracellular pH, in heart disease, and in cytoskeletal organization. The first 500 amino acids of the protein are believed to consist of 12 transmembrane helices, a membrane-associated segment, and two reentrant loops. A C-terminal regulatory domain of approximately 315 amino acids regulates the protein and mediates cytoskeletal interactions. Studies are underway to determine the amino acid residues important in NHE1 function. At present, it is clear that transmembrane segment IV is important in NHE1 function and that transmembrane segments VII and IX are also involved in transport. Further experiments are required to elucidate the mechanism of transport and regulation of this multifunctional protein.     

The Na+/H+ exchanger isoform 1

The Na+/H+ exchanger (NHE) isoform 1 is a ubiquitously expressed integral membrane protein which regulates intracellular pH in mammalian cells. Nine isoforms of the Na+/H+ exchanger have been identified. The isoform first discovered has two domains: an N-terminal membrane domain containing approximately 500 amino acids and a C-terminal regulatory domain containing approximately 315 amino acids. The exchanger, which resides in the plasma membrane, exchanges an intracellular proton for an extracellular sodium, thereby regulating intracellular pH. It is involved in cell growth and differentiation, cell migration, and regulation of sodium fluxes. The Na+/H+ exchanger plays an important role in myocardial damage during ischemia and reperfusion and has recently been implicated as a mediator of cardiac hypertrophy. Inhibitors of the Na+/H+ exchanger, which may prove useful in the clinical treatment of these conditions, are currently being developed and clinical trials are underway.     

Integrin-mediated membrane blebbing is dependent on sodium-proton exchanger 1 and sodium-calcium exchanger 1 activity

Integrin signaling and membrane blebbing modulate cell adhesion, spreading, and migration. However, the relationship between integrin signaling and membrane blebbing is unclear. Here, we show that an integrin-ligand interaction induces both membrane blebbing and changes in membrane permeability. Sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are membrane proteins located on the bleb membrane. Inhibition of NHE1 disrupts membrane blebbing and decreases changes in membrane permeability. However, inhibition of NCX1 enhances cell blebbing; cells become swollen because of NHE1 induced intracellular sodium accumulation. Our study found that NHE1 induced sodium influx is a driving force for membrane bleb growth, while sodium efflux (and calcium influx) induced by NCX1 in a reverse mode results in membrane bleb retraction. Together, these findings reveal a novel function for NHE1 and NCX1 in membrane blebbing and permeability, and establish a link between membrane blebbing and integrin signaling.     

Unconventional Chemiosmotic Coupling of NHA2, a Mammalian Na+/H+ Antiporter,to a Plasma Membrane H+ Gradient

Human NHA2, a newly discovered cation proton antiporter, is implicated in essential hypertension by gene linkage analysis. We show that NHA2 mediates phloretin-sensitive Na⁺-Li⁺ counter-transport (SLC) activity, an established marker for hypertension. In contrast to bacteria and fungi where H⁺ gradients drive uptake of metabolites, secondary transport at the plasma membrane of mammalian cells is coupled to the Na⁺ electrochemical gradient. Our findings challenge this paradigm by showing coupling of NHA2 and V-type H⁺-ATPase at the plasma membrane of kidney-derived MDCK cells, resulting in a virtual Na⁺ efflux pump. Thus, NHA2 functionally recapitulates an ancient shared evolutionary origin with bacterial NhaA. Although plasma membrane H⁺ gradients have been observed in some specialized mammalian cells, the ubiquitous tissue distribution of NHA2 suggests that H⁺-coupled transport is more widespread. The coexistence of Na⁺ and H⁺-driven chemiosmotic circuits has implications for salt and pH regulation in the kidney.     

Regulation of mitogen-activated protein kinase pathways by the plasma membrane Na+/H+ exchanger, NHE1

The mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, play a major role in the regulation of pivotal cellular processes such as cell death/survival balance, cell cycle progression, and cell migration. MAPK activity is regulated by a three-tiered phosphorelay system, which is in turn regulated by a complex network of signaling events and scaffolding proteins. The ubiquitous plasma membrane Na(+)/H(+) exchanger NHE1 is activated by, and implicated in, the physiological/pathophysiological responses to many of the same stimuli that modulate MAPK activity. While under some conditions, NHE1 is regulated by MAPKs, a number of studies have, conversely, implicated NHE1 in the regulation of MAPK activity. Here, we discuss the current evidence indicating the involvement of NHE1 in MAPK regulation, the mechanisms by which this may occur, and the possible physiological and pathophysiological relevance of this phenomenon.     

Characterization of cytoskeletal protein 4.1R interaction with NHE1 (Na+/H+ exchanger isoform 1)

NHE1 (Na+/H+ exchanger isoform 1) has been reported to be hyperactive in 4.1R-null erythrocytes [Rivera, De Franceschi, Peters, Gascard, Mohandas and Brugnara (2006) Am. J. Physiol. Cell Physiol. 291, C880-C886], supporting a functional interaction between NHE1 and 4.1R. In the present paper we demonstrate that 4.1R binds directly to the NHE1cd (cytoplasmic domain of NHE1) through the interaction of an EED motif in the 4.1R FERM (4.1/ezrin/radixin/moesin) domain with two clusters of basic amino acids in the NHE1cd, K519R and R556FNKKYVKK, previously shown to mediate PIP2 (phosphatidylinositol 4,5-bisphosphate) binding [Aharonovitz, Zaun, Balla, York, Orlowski and Grinstein (2000) J. Cell. Biol. 150, 213-224]. The affinity of this interaction (Kd=100-200?nM) is reduced in hypertonic and acidic conditions, demonstrating that this interaction is of an electrostatic nature. The binding affinity is also reduced upon binding of Ca2+/CaM (Ca2+-saturated calmodulin) to the 4.1R FERM domain. We propose that 4.1R regulates NHE1 activity through a direct protein-protein interaction that can be modulated by intracellular pH and Na+ and Ca2+ concentrations.     

Functional characterization of the transmembrane segment VII of the NHE1 isoform of the Na+/H+ exchanger

The Na+/H+ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH. It extrudes 1 intracellular H+ in exchange for 1 extracellular Na+. It has 2 large domains, an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the critical transmembrane segment TM VII. Residues Leu 255, Leu 258, Glu 262, Leu 265, Asn 266, Asp 267, Val 269, Val 272, and Leu 273 were all mutated to cysteine residues in the cysteineless NHE1 isoform. Mutation of amino acids E262, N266, and D267 caused severe defects in activity and targeting of the intact full length protein. The balance of the active mutants were examined for sensitivity to the sulfhydryl reactive reagents, positively charged MTSET ((2- (trimethylammonium)ethyl)methanethiosulfonate) and negatively charged MTSES ((2-sulfonatoethyl)methanethiosulfonate). Leu 255 and Leu 258 were sensitive to MTSET but not to MTSES. The results suggest that these amino acids are pore-lining residues. We present a model of TM VII that shows that residues Leu 255, Leu 258, Glu 262, Asn 266, and Asp 267 lie near the same face of TM VII, lining the ion transduction pore.     

Expression and characterization of the Na+/H+ exchanger in the mammalian myocardium

We examined two expression systems for studying the Na⁺/H⁺ exchanger in the mammalian myocardium. Mammalian NHE1 with a hemagglutinin (HA) tag and was cloned behind the alpha myosin heavy chain promoter. Transgenic mice were made with wild type NHE1 protein or with a hyperactive NHE1 protein mutated at the calmodulin-binding domain. Three lines of transgenic mice were made of each cDNA with expression levels of each type varying from high to low. Higher levels and activity of the Na⁺/H⁺ exchanger were associated with decreased long-term survival of mice, and with dilated or hypertrophic cardiomyopathy. The exogenous NHE1 protein was present in freshly made cardiomyocytes from transgenic mice, however, expression from the alpha myosin heavy chain promoter declined rapidly and little exogenous NHE1 was apparent on the fourth day after cardiomyocyte isolation. To express NHE1 protein in isolated cardiomyocytes, we transferred a mutated form of the protein into an adenoviral expression system. Infection of neonatal rat cardiomyocytes resulted in robust expression of the exogenous NHE1 protein. The mutant form of the NHE1 protein could be distinguished from the endogenous Na⁺/H⁺ exchanger by its resistance to inhibition by amiloride analogs. Our results suggest that for in vivo studies on intact hearts and animals, expression in transgenic mice is an appropriate system, however for long-term studies on cardiomyocytes, this model is inappropriate due to waning expression from the alpha myosin heavy chain promoter. Therefore, infection by adenovirus is a superior system for long-term studies on cardiomyocytes in culture.     

Structural and functional characterization of transmembrane segment IX of the NHE1 isoform of the Na+/H+ exchanger

The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by removing one intracellular H(+) in exchange for one extracellular Na(+). It has a large N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the putative transmembrane segment IX (residues 339-363). Each residue was mutated to cysteine in a functional cysteineless NHE1 protein. Of 25 amino acids mutated, 5 were inactive or nearly so after mutation to cysteine. Several of these showed aberrant targeting to the plasma membrane and reduced expression of the intact protein, whereas others were expressed and targeted correctly but had defective NHE1 function. Of the active mutants, Glu(346) and Ser(351) were inhibited >70% by positively charged [2-(trimethylammonium)-ethyl]methanethiosulfonate but not by anionic [2-sulfonatoethyl]methanethiosulfonate, suggesting that they are pore lining and make up part of the cation conduction pathway. Both mutants also had decreased affinity for Na(+) and decreased activation by intracellular protons. The structure of a peptide representing amino acids 338-365 was determined by using high resolution NMR in dodecylphosphocholine micelles. The structure contained two helical regions (amino acids Met(340)-Ser(344) and Ile(353)-Ser(359)) kinked with a large bend angle around a pivot point at amino acid Ser(351). The results suggest that transmembrane IX is critical with pore-lining residues and a kink at the functionally important residue Ser(351).