Subunit δ Is the Key Player for Assembly of the H+-translocating Unit of Escherichia coli FOF1 ATP Synthase

The ATP synthase (F_OF₁) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane to the synthesis or hydrolysis of ATP. This nanomotor is composed of the rotor c₁₀γϵ and the stator ab₂α₃β₃δ. To study the assembly of this multimeric enzyme complex consisting of membrane-integral as well as peripheral hydrophilic subunits, we combined nearest neighbor analyses by intermolecular disulfide bond formation or purification of partially assembled F_OF₁ complexes by affinity chromatography with the use of mutants synthesizing different sets of F_OF₁ subunits. Together with a time-delayed in vivo assembly system, the results demonstrate that F_OF₁ is assembled in a modular way via subcomplexes, thereby preventing the formation of a functional H⁺-translocating unit as intermediate product. Surprisingly, during the biogenesis of F_OF₁, F₁ subunit δ is the key player in generating stable F_O. Subunit δ serves as clamp between ab₂ and c₁₀α₃β₃γϵ and guarantees that the open H⁺ channel is concomitantly assembled within coupled F_OF₁ to maintain the low membrane proton permeability essential for viability, a general prerequisite for the assembly of multimeric H⁺-translocating enzymes.     

Composition of the central stalk of the Na+-pumping V-ATPase from Caloramator fervidus

The Na⁺-pumping V-ATPase complex of the thermophilic bacterium Caloramator fervidus was purified and dissociated under controlled conditions. The structure of purified V₁-ATPase subcomplexes differing in subunit composition was analyzed by electron microscopy and single particle analysis of 50 000 projections. Difference mapping of subcomplex projections revealed the presence and position of two subunits in the central stalk. A density with an elongated shape similar to the γ subunit of F-ATPases is partly located within V₁ and corresponds, most likely, to subunit E. Subunit E is connected to the membrane-bound part V₀ via subunit C, a spherical density that is connected to the center of V₀. The presence of subunit C makes the central stalk substantially longer in comparison to the F-ATPases, in which the γ subunit connects directly to F₀.     

Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F1-ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

F₁-ATPase is a rotary molecular machine with a subunit stoichiometry of α₃β₃γ₁δ₁ε₁. It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α₃β₃ core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F₁-ATPase have suggested that the α₃β₃ core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F₁-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ^o) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F₁ complexes retained 21.7 to 44.6% of the native F₁-ATPase activity. The γ-less F₁ subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ^o conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α₃β₃ subcomplex.     

The Ca2+ channel β2 subunit is selectively targeted to the axon terminals of supraoptic neurons

The assembly of high voltage-activated Ca²⁺ channels with different β subunits influences channel properties and possibly subcellular targeting. We studied β subunit expression in the somata and axon terminals of the magnocellular neurosecretory cells, which are located in the supraoptic nucleus (SON) and neurohypophysis, respectively. Antibodies directed against the 4 Ca_Vβ subunits (Ca_Vβ₁-Ca_Vβ₄) were used for immunoblots and for immunostaining of slices of these two tissues. We found that all 4 β subunits are expressed in both locations, but that Ca_Vβ₂ had the highest relative expression in the neurohypophysis. These data suggest that the Ca_Vβ₂ subunit is selectively targeted to axon terminals and may play a role in targeting and/or regulating the properties of Ca²⁺ channels.     

Diversity of Channels Generated by Different Combinations of Epithelial Sodium Channel Subunits

The epithelial sodium channel is a multimeric protein formed by three homologous subunits: α, β, and γ; each subunit contains only two transmembrane domains. The level of expression of each of the subunits is markedly different in various Na⁺ absorbing epithelia raising the possibility that channels with different subunit composition can function in vivo. We have examined the functional properties of channels formed by the association of α with β and of α with γ in the Xenopus oocyte expression system using two-microelectrode voltage clamp and patch-clamp techniques. We found that αβ channels differ from αγ channels in the following functional properties: (a) αβ channels expressed larger Na⁺ than Li⁺ currents (I_Na+/I_Li+ 1.2) whereas αγ channels expressed smaller Na⁺ than Li⁺ currents (I_Na+/I_Li+ 0.55); (b) the Michaelis Menten constants (K _m) of activation of current by increasing concentrations of external Na⁺ and Li⁺ of αβ channels were larger (K _m > 180 mM) than those of αγ channels (K _m of 35 and 50 mM, respectively); (c) single channel conductances of αβ channels (5.1 pS for Na⁺ and 4.2 pS for Li⁺) were smaller than those of αγ channels (6.5 pS for Na⁺ and 10.8 pS for Li⁺); (d) the half-inhibition constant (K _i) of amiloride was 20-fold larger for αβ channels than for αγ channels whereas the K _i of guanidinium was equal for both αβ and αγ. To identify the domains in the channel subunits involved in amiloride binding, we constructed several chimeras that contained the amino terminus of the γ subunit and the carboxy terminus of the β subunit. A stretch of 15 amino acids, immediately before the second transmembrane domain of the β subunit, was identified as the domain conferring lower amiloride affinity to the αβ channels. We provide evidence for the existence of two distinct binding sites for the amiloride molecule: one for the guanidium moiety and another for the pyrazine ring. At least two subunits α with β or γ contribute to these binding sites. Finally, we show that the most likely stoichiometry of αβ and αγ channels is 1α:1β and 1α:1γ, respectively.     

Architecture of the eIF2B regulatory subcomplex and its implications for the regulation of guanine nucleotide exchange on eIF2

Eukaryal translation initiation factor 2B (eIF2B) acts as guanine nucleotide exchange factor (GEF) for eIF2 and forms a central target for pathways regulating global protein synthesis. eIF2B consists of five non-identical subunits (α–ϵ), which assemble into a catalytic subcomplex (γ, ϵ) responsible for the GEF activity, and a regulatory subcomplex (α, β, δ) which regulates the GEF activity under stress conditions. Here, we provide new structural and functional insight into the regulatory subcomplex of eIF2B (eIF2B^RSC). We report the crystal structures of eIF2Bβ and eIF2Bδ from Chaetomium thermophilum as well as the crystal structure of their tetrameric eIF2B(βδ)₂ complex. Combined with mutational and biochemical data, we show that eIF2B^RSC exists as a hexamer in solution, consisting of two eIF2Bβδ heterodimers and one eIF2Bα₂ homodimer, which is homologous to homohexameric ribose 1,5-bisphosphate isomerases. This homology is further substantiated by the finding that eIF2Bα specifically binds AMP and GMP as ligands. Based on our data, we propose a model for eIF2B^RSC and its interactions with eIF2 that is consistent with previous biochemical and genetic data and provides a framework to better understand eIF2B function, the molecular basis for Gcn⁻, Gcd⁻ and VWM/CACH mutations and the evolutionary history of the eIF2B complex.     

Thermodynamic Analyses of Nucleotide Binding to an Isolated Monomeric β Subunit and the α3β3γ Subcomplex of F1-ATPase

Rotation of the γ subunit of the F₁-ATPase plays an essential role in energy transduction by F₁-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F₁-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α₃β₃γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K_d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH⁰ and ΔG⁰ values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α₃β(Y341W)₃γ subcomplex had similar K_d and ΔG⁰ values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.     

Major Binding Sites for the Nuclear Import Receptor Are the Internal Nucleoporin Nup153 and the Adjacent Nuclear Filament Protein Tpr

A major question in nuclear import concerns the identity of the nucleoporin(s) that interact with the nuclear localization sequences (NLS) receptor and its cargo as they traverse the nuclear pore. Ligand blotting and solution binding studies of isolated proteins have attempted to gain clues to the identities of these nucleoporins, but the studies have from necessity probed binding events far from an in vivo context. Here we have asked what binding events occur in the more physiological context of a Xenopus egg extract, which contains nuclear pore subcomplexes in an assembly competent state. We have then assessed our conclusions in the context of assembled nuclear pores themselves. We have used immunoprecipitation to identify physiologically relevant complexes of nucleoporins and importin subunits. In parallel, we have demonstrated that it is possible to obtain immunofluorescence localization of nucleoporins to subregions of the nuclear pore and its associated structures. By immunoprecipitation, we find the nucleoporin Nup153 and the pore-associated filament protein Tpr, previously shown to reside at distinct sites on the intranuclear side of assembled pores, are each in stable subcomplexes with importin α and β in Xenopus egg extracts. Importin subunits are not in stable complexes with nucleoporins Nup62, Nup93, Nup98, or Nup214/CAN, either in egg extracts or in extracts of assembled nuclear pores. In characterizing the Nup153 complex, we find that Nup153 can bind to a complete import complex containing importin α, β, and an NLS substrate, consistent with an involvement of this nucleoporin in a terminal step of nuclear import. Importin β binds directly to Nup153 and in vitro can do so at multiple sites in the Nup153 FXFG repeat region. Tpr, which has no FXFG repeats, binds to importin β and to importin α/β heterodimers, but only to those that do not carry an NLS substrate. That the complex of Tpr with importin β is fundamentally different from that of Nup153 is additionally demonstrated by the finding that recombinant β or β_45–462 fragment freely exchanges with the endogenous importin β/Nup153 complex, but cannot displace endogenous importin β from a Tpr complex. However, the GTP analogue GMP-PNP is able to disassemble both Nup153– and Tpr–importin β complexes. Importantly, analysis of extracts of isolated nuclei indicates that Nup153– and Tpr–importin β complexes exist in assembled nuclear pores. Thus, Nup153 and Tpr are major physiological binding sites for importin β. Models for the roles of these interactions are discussed.     

The β-Prism Lectin Domain of Vibrio cholerae Hemolysin Promotes Self-assembly of the β-Pore-forming Toxin by a Carbohydrate-independent Mechanism

Vibrio cholerae cytolysin/hemolysin (VCC) is an amphipathic 65-kDa β-pore-forming toxin with a C-terminal β-prism lectin domain. Because deletion or point mutation of the lectin domain seriously compromises hemolytic activity, it is thought that carbohydrate-dependent interactions play a critical role in membrane targeting of VCC. To delineate the contributions of the cytolysin and lectin domains in pore formation, we used wild-type VCC, 50-kDa VCC (VCC⁵⁰) without the lectin domain, and mutant VCC^D617A with no carbohydrate-binding activity. VCC and its two variants with no carbohydrate-binding activity moved to the erythrocyte stroma with apparent association constants on the order of 10⁷ m⁻¹. However, loss of the lectin domain severely reduced the efficiency of self-association of the VCC monomer with the β-barrel heptamer in the synthetic lipid bilayer from ∼83 to 27%. Notably, inactivation of the carbohydrate-binding activity by the D617A mutation marginally reduced oligomerization to ∼77%. Oligomerization of VCC⁵⁰ was temperature-insensitive; by contrast, VCC self-assembly increased with increasing temperature, suggesting that the process is driven by entropy and opposed by enthalpy. Asialofetuin, the β₁-galactosyl-terminated glycoprotein inhibitor of VCC-induced hemolysis, promoted oligomerization of 65-kDa VCC to a species that resembled the membrane-inserted heptamer in stoichiometry and morphology but had reduced global amphipathicity. In conclusion, we propose (i) that the β-prism lectin domain facilitated toxin assembly by producing entropy during relocation in the heptamer and (ii) that glycoconjugates inhibited VCC by promoting its assembly to a water-soluble, less amphipathic oligomer variant with reduced ability to penetrate the bilayer.     

Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds

The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the α^A-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the α^D-globin gene). The α^D-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O₂ affinity in the presence of allosteric effectors such as organic phosphates and Cl⁻ ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O₂ affinity stems primarily from changes in the O₂ association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the α^D-globin gene that is shared with the embryonic α-like globin gene.     

Nicotinic Receptor Subunit α5 Modifies Assembly,Up-regulation,and Response to Pro-inflammatory Cytokines

In the mammalian brain high affinity nicotine-binding sites are composed of at least the α4 and β2 subunits. Additional nicotinic acetylcholine receptor subunits that are often co-expressed with α4+β2 include α5. The introduction of α5 into 293 cells expressing α4+β2 strongly favors assembly of α4+α5+β2 receptors, increases constitutive ligand binding density as measured using [³H]epibatidine, but reduces the magnitude of up-regulation in response to chronic nicotine. In contrast, when β4 is substituted for β2, α5 interferes with the assembly of these receptors, demonstrating an important role for the β subunit in this process. When cells co-express α4+α5+β2+β4, over 50% of the subunit associations include all four subunits, but they fail to be detected using [³H]epibatidine binding. However, complexes of α4+α5+β2 do preferentially emerge from these subunit mixtures, and these mixtures bind ligand. In previous studies of α4+β2+β4 co-expression by 293 cells, the inflammatory cytokines IL-1β and TNFα influenced the outcome of receptor assembly (Gahring, L. C., Days, E. L., Kaasch, T., González de Mendoza, M., Owen, L., Persiyanov, K., and Rogers, S. W. (2005) J. Neuroimmunol. 166, 88–101). When α5 is included in this subunit mixture, and cells are exposed to either inflammatory cytokine, subunit association is no longer altered. These findings suggest that α5 is an influential modulator of α4+β2 nicotinic acetylcholine receptor assembly and stabilizes their expression in response to fluctuations in external conditions.     

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)₄ complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)₂ lobes joined with D₂ symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)₄ complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)₄ kinase core, because a β₄ subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit.     

Characteristics of an R-Phycoerythrin with Two γ Subunits Prepared from Red Macroalga Polysiphonia urceolata

An R-phycoerythrin (R-PE) was isolated by gel filtrations on Sepharose CL-4B and Sephadex G-150 from the phycobiliprotein extract of the marine red macroalga Polysiphonia urceolata Grev and further purified by ion exchange chromatography on DEAE-Sepharose Fast Flow. The purified R-PE showed three absorption peaks at 498 nm, 538 nm, 566 nm and one fluorescent emission maximum at 577 nm. Although the R-PE showed a single band on the examination by native PAGE, it exhibited two very close bands at pH about 4.7 in native isoelectric focusing (IEF). Polypeptide analysis of the R-PE demonstrated that it contained four chromophore-carrying subunits, α^18.2, β^20.6, γ^31.6 (γ''), γ^34.6 (γ), and no colorless polypeptide; its subunit composition was 6α^18.2:6β^20.6:1 γ^31.6:2γ^34.6. The α and β subunits were distributed within a acidic pH range from 5.0 to 6.0 in denaturing IEF and the γ subunits were in a basic pH range from 7.6 to 8.1. These results reveal that the prepared R-PE may exist in two hexamers of γ (αβ)₃ γ (αβ)₃γ'' and γ (αβ)₃ γ''(αβ)₃ γ and that the R-PE participate in the rod domain assembly of P. urceolata phycobilisomes by stacking each of its trimer (αβ)₃ face-to-face with the aid of one γ subunit (γ or γ'').     

Mechanism of Auxiliary Subunit Modulation of Neuronal α1E Calcium Channels

Voltage-gated calcium channels are composed of a main pore-forming α₁ moiety, and one or more auxiliary subunits (β, α₂δ) that modulate channel properties. Because modulatory properties may vary greatly with different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of α_1E calcium channels in combination with a β (β₁–β₄) and/or the α₂δ subunit, exploiting both ionic- and gating-current measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting α₂δ with β subunits, of transfecting two different β subunits simultaneously, and of COOH-terminal truncation of α_1E to remove a second β binding site. The main results are as follows. (a) The α₂δ and β subunits modulate α_1E in fundamentally different ways. The sole effect of α₂δ is to increase current density by elevating channel density. By contrast, though β subunits also increase functional channel number, they also enhance maximum open probability (G_max/Q_max) and hyperpolarize the voltage dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation kinetics. Different β isoforms produce nearly indistinguishable effects on activation. However, β subunits produced clear, isoform-specific effects on inactivation properties. (b) All the β subunit effects can be explained by a gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no clear evidence for simultaneous modulation by two different β subunits. (d) The modulatory features found here for α_1E do not generalize uniformly to other α₁ channel types, as α_1C activation gating shows marked β isoform dependence that is absent for α_1E. Together, these results help to establish a more comprehensive picture of auxiliary-subunit regulation of α_1E calcium channels.     

Subunit Composition Determines the Single Channel Kinetics of the Epithelial Sodium Channel

We have further characterized at the single channel level the properties of epithelial sodium channels formed by coexpression of α with either wild-type β or γ subunits and α with carboxy-terminal truncated β (β_T) or γ (γ_T) subunits in Xenopus laevis oocytes. αβ and αβ_T channels (9.6 and 8.7 pS, respectively, with 150 mM Li⁺) were found to be constitutively open. Only upon inclusion of 1 μM amiloride in the pipette solution could channel activity be resolved; both channel types had short open and closed times. Mean channel open probability (P _o) for αβ was 0.54 and for αβ_T was 0.50. In comparison, αγ and αγ_T channels exhibited different kinetics: αγ channels (6.7 pS in Li⁺) had either long open times with short closings, resulting in a high P _o (0.78), or short openings with long closed times, resulting in a low P _o (0.16). The mean P _o for all αγ channels was 0.48. αγ_T (6.6 pS in Li⁺) behaved as a single population of channels with distinct kinetics: mean open time of 1.2 s and closed time of 0.4 s, with a mean P _o of 0.6, similar to that of αγ. Inclusion of 0.1 μM amiloride in the pipette solution reduced the mean open time of αγ_T to 151 ms without significantly altering the closed time. We also examined the kinetics of amiloride block of αβ, αβ_T (1 μM amiloride), and αγ_T (0.1 μM amiloride) channels. αβ and αβ_T had similar blocking and unblocking rate constants, whereas the unblocking rate constant for αγ_T was 10-fold slower than αβ_T. Our results indicate that subunit composition of ENaC is a main determinant of P _o. In addition, channel kinetics and P _o are not altered by carboxy-terminal deletion in the β subunit, whereas a similar deletion in the γ subunit affects channel kinetics but not P _o.     

Carbon Dioxide is a Powerful Inducer of Monokaryotic Hyphae and Spore Development in Cryptococcus gattii and Carbonic Anhydrase Activity is Dispensable in This Dimorphic Transition

Cryptococcus gattii is unique among human pathogenic fungi with specialized ecological niche on trees. Since leaves concentrate CO₂, we investigated the role of this gaseous molecule in C. gattii biology and virulence. We focused on the genetic analyses of β-carbonic anhydrase (β-CA) encoded by C. gattii CAN1 and CAN2 as later is critical for CO₂ sensing in a closely related pathogen C. neoformans. High CO₂ conditions induced robust development of monokaryotic hyphae and spores in C. gattii. Conversely, high CO₂ completely repressed hyphae development in sexual mating. Both CAN1 and CAN2 were dispensable for CO₂ induced morphogenetic transitions. However, C. gattii CAN2 was essential for growth in ambient air similar to its reported role in C. neoformans. Both can1 and can2 mutants retained full pathogenic potential in vitro and in vivo. These results provide insight into C. gattii adaptation for arboreal growth and production of infectious propagules by β-CA independent mechanism(s).     

Identification of Amino Acid Residues in the α, β, and γ Subunits of the Epithelial Sodium Channel (ENaC) Involved in Amiloride Block and Ion Permeation

The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three αβγ subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in β and γ subunits at position βG525 and γG537 increased the apparent inhibitory constant (K _i) for amiloride by >1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the α subunit increased amiloride K _i by 20-fold, without changing channel conducting properties. Coexpression of these mutated αβγ subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn²⁺ ions, in particular the αS583C mutant showing a K _i for Zn²⁺of 29 μM. Mutations of residues αW582L or βG522D also increased amiloride K _i, the later mutation generating a Ca²⁺blocking site located 15% within the membrane electric field. These experiments provide strong evidence that αβγ ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of αβγ subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na⁺ions at an external Na⁺binding site preventing ion permeation through the channel pore.