A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers

AQPs (aquaporins) are conserved in all kingdoms of life and facilitate the rapid diffusion of water and/or other small solutes across cell membranes. Among the different plant AQPs, PIPs (plasma membrane intrinsic proteins), which fall into two phylogenetic groups, PIP1 and PIP2, play key roles in plant water transport processes. PIPs form tetramers in which each monomer acts as a functional channel. The intermolecular interactions that stabilize PIP oligomer complexes and are responsible for the resistance of PIP dimers to denaturating conditions are not well characterized. In the present study, we identified a highly conserved cysteine residue in loop A of PIP1 and PIP2 proteins and demonstrated by mutagenesis that it is involved in the formation of a disulfide bond between two monomers. Although this cysteine seems not to be involved in regulation of trafficking to the plasma membrane, activity, substrate selectivity or oxidative gating of ZmPIP1s (Zm is Zea mays), ZmPIP2s and hetero-oligomers, it increases oligomer stability under denaturating conditions. In addition, when PIP1 and PIP2 are co-expressed, the loop A cysteine of ZmPIP1;2, but not that of ZmPIP2;5, is involved in the mercury sensitivity of the channels.     

Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6

The molecular assembly of the epithelial Ca(2+) channels (TRPV5 and TRPV6) was investigated to determine the subunit stoichiometry and composition. Immunoblot analysis of Xenopus laevis oocytes expressing TRPV5 and TRPV6 revealed two specific bands of 75 and 85-100 kDa, corresponding to the core and glycosylated proteins, respectively, for each channel. Subsequently, membranes of these oocytes were sedimented on sucrose gradients. Immuno blotting revealed that TRPV5 and TRPV6 complexes migrate with a mol. wt of 400 kDa, in line with a tetrameric structure. The tetrameric stoichiometry was confirmed in an electrophysiological analysis of HEK293 cells co-expressing concatemeric channels together with a TRPV5 pore mutant that reduced Cd(2+) sensitivity and voltage-dependent gating. Immuno precipitations using membrane fractions from oocytes co-expressing TRPV5 and TRPV6 demonstrated that both channels can form heteromeric complexes. Expression of all possible heterotetrameric TRPV5/6 complexes in HEK293 cells resulted in Ca(2+) channels that varied with respect to Ca(2+)-dependent inactivation, Ba(2+) selectivity and pharmacological block. Thus, Ca(2+)-transporting epithelia co-expressing TRPV5 and TRPV6 can generate a pleiotropic set of functional heterotetrameric channels with different Ca(2+) transport kinetics.     

The oligomeric state of the active BM2 ion channel protein of influenza B virus

Influenza A virus and influenza B virus particles both contain small integral membrane proteins (A/M2 and BM2, respectively) that function as a pH-sensitive proton channel and are essential for virus replication. The mechanism of action of the M2 channels is a subject of scientific interest particularly as A/M2 channel was shown to be a target for the action of the antiviral drug amantadine. Unfortunately, an inhibitor of the BM2 channel activity is not known. Thus, knowledge of the structural and functional properties of the BM2 channel is essential for the development of potent antiviral drugs. The characterization of the oligomeric state of the BM2 channel is an essential first step in the understanding of channel function. Here we describe determination of the stoichiometry of the BM2 proton channel by utilizing three different approaches. 1) We demonstrated that BM2 monomers can be chemically cross-linked to yield species consistent with dimers, trimers, and tetramers. 2) We studied electrophysiological and biochemical properties of mixed oligomers consisting of wild-type and mutated BM2 subunits and related these data to predicted binomial distribution models. 3) We used fluorescence resonance energy transfer (FRET) in combination with biochemical measurements to estimate the relationships between BM2 channel subunits expressed in the plasma membrane. Our experimental data are consistent with a tetrameric structure of the BM2 channel. Finally, we demonstrated that BM2 transmembrane domain is responsible for the channel oligomerization.     

Assembly stoichiometry of the GluK2/GluK5 kainate receptor complex

Ionotropic glutamate receptors assemble as homo- or heterotetramers. One well-studied heteromeric complex is formed by the kainate receptor subunits GluK2 and GluK5. Retention motifs prevent trafficking of GluK5 homomers to the plasma membrane, but coassembly with GluK2 yields functional heteromeric receptors. Additional control over GluK2/GluK5 assembly seems to be exerted by the aminoterminal domains, which preferentially assemble into heterodimers as isolated domains. However,the stoichiometry of the full-length GluK2/GluK5 receptor complex has yet to be determined, as is the case for all non-NMDA glutamate receptors. Here, we address this question, using a single-molecule imaging technique that enables direct counting of the number of each GluK subunit type in homomeric and heteromeric receptors in the plasma membranes of live cells. We show that GluK2 and GluK5 assemble with 2:2 stoichiometry. This is an important step toward understanding the assembly mechanism, architecture, and functional consequences of heteromer formation in ionotropic glutamate receptors.     

Single-molecule analysis of PIP2;1 dynamics and partitioning reveals multiple modes of Arabidopsis plasma membrane aquaporin regulation

PIP2;1 is an integral membrane protein that facilitates water transport across plasma membranes. To address the dynamics of Arabidopsis thaliana PIP2;1 at the single-molecule level as well as their role in PIP2;1 regulation, we tracked green fluorescent protein-PIP2;1 molecules by variable-angle evanescent wave microscopy and fluorescence correlation spectroscopy (FCS). Single-particle tracking analysis revealed that PIP2;1 presented four diffusion modes with large dispersion of diffusion coefficients, suggesting that partitioning and dynamics of PIP2;1 are heterogeneous and, more importantly, that PIP2;1 can move into or out of membrane microdomains. In response to salt stress, the diffusion coefficients and percentage of restricted diffusion increased, implying that PIP2;1 internalization was enhanced. This was further supported by the decrease in PIP2;1 density on plasma membranes by FCS. We additionally demonstrated that PIP2;1 internalization involves a combination of two pathways: a tyrphostin A23-sensitive clathrin-dependent pathway and a methyl-β-cyclodextrin-sensitive, membrane raft-associated pathway. The latter was efficiently stimulated under NaCl conditions. Taken together, our findings demonstrate that PIP2;1 molecules are heterogeneously distributed on the plasma membrane and that clathrin and membrane raft pathways cooperate to mediate the subcellular trafficking of PIP2;1, suggesting that the dynamic partitioning and recycling pathways might be involved in the multiple modes of regulating water permeability.     

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Phosphatidylinositol bisphosphate (PIP(2)) is the most abundant phosphoinositide in the plasma membrane of cells and its interaction with many ion channel proteins has proven to be a critical factor enabling ion channel gating. All members of the inwardly rectifying potassium (Kir) channel family depend on PIP(2) for their activity, displaying distinct affinities and stereospecificities of interaction with the phosphoinositide. Here, we explored the stoichiometry of Kir channels with PIP(2). We first showed that PIP(2) regulated the activity of Kir3.4 channels mainly by altering their bursting behavior. Detailed burst analysis indicates that the channels assumed up to four open states and a connectivity of four between open and closed states depending on the available PIP(2) levels. Moreover, by controlling the number of PIP(2)-sensitive subunits in the stoichiometry of a tetrameric Kir2.1 channel, we showed that characteristic channel activity was obtained when at least two wild-type subunits were present. Our studies support a kinetic model for gating of Kir channels by PIP(2), where each of the four open states corresponds to the channel activated by one to four PIP(2) molecules.     

Pyridoxal phosphate inhibits dynamic subunit interchange among serine hydroxymethyltransferase tetramers

Cytoplasmic serine hydroxymethyltransferase (cSHMT) is a tetrameric, pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. The enzyme has four active sites and is best described as a dimer of obligate dimers. Each monomeric subunit within the obligate dimer contributes catalytically important amino acid residues to both active sites. To investigate the interchange of subunits among cSHMT tetramers, a dominant-negative human cSHMT enzyme (DNcSHMT) was engineered by making three amino acid substitutions: K257Q, Y82A, and Y83F. Purified recombinant DNcSHMT protein was catalytically inactive and did not bind 5-formyltetrahydrofolate. Coexpression of the cSHMT and DNcSHMT proteins in bacteria resulted in the formation of heterotetramers with a cSHMT/DNcSHMT subunit ratio of 1. Characterization of the cSHMT/DNcSHMT heterotetramers indicates that DNcSHMT and cSHMT monomers randomly associate to form tetramers and that cSHMT/DNcSHMT obligate dimers are catalytically inactive. Incubation of recombinant cSHMT protein with recombinant DNcSHMT protein did not result in the formation of hetero-oligomers, indicating that cSHMT subunits do not exchange once the tetramer is assembled. However, removal of the active site PLP cofactor does permit exchange of obligate dimers among preformed cSHMT and DNcSHMT tetramers, and the formation of heterotetramers from cSHMT and DNcSHMT homodimers does not affect the activity of the cSHMT homodimers. The results of these studies demonstrate that PLP inhibits dimer exchange among cSHMT tetramers and suggests that cellular PLP concentrations may influence the stability of cSHMT protein in vivo.     

Conserved Negative Charges in the N-terminal Tetramerization Domain Mediate Efficient Assembly of Kv2.1 and Kv2.1/Kv6.4 Channels

Voltage-gated potassium (Kv) channels are transmembrane tetramers of individual α-subunits. Eight different Shaker-related Kv subfamilies have been identified in which the tetramerization domain T1, located on the intracellular N terminus, facilitates and controls the assembly of both homo- and heterotetrameric channels. Only the Kv2 α-subunits are able to form heterotetramers with members of the silent Kv subfamilies (Kv5, Kv6, Kv8, and Kv9). The T1 domain contains two subdomains, A and B box, which presumably determine subfamily specificity by preventing incompatible subunits to assemble. In contrast, little is known about the involvement of the A/B linker sequence. Both Kv2 and silent Kv subfamilies contain a fully conserved and negatively charged sequence (CDD) in this linker that is lacking in the other subfamilies. Neutralizing these aspartates in Kv2.1 by mutating them to alanines did not affect the gating properties, but reduced the current density moderately. However, charge reversal arginine substitutions strongly reduced the current density of these homotetrameric mutant Kv2.1 channels and immunocytochemistry confirmed the reduced expression at the plasma membrane. Förster resonance energy transfer measurements using confocal microscopy showed that the latter was not due to impaired trafficking, but to a failure to assemble the tetramer. This was further confirmed with co-immunoprecipitation experiments. The corresponding arginine substitution in Kv6.4 prevented its heterotetrameric interaction with Kv2.1. These results indicate that these aspartates (especially the first one) in the A/B box linker of the T1 domain are required for efficient assembly of both homotetrameric Kv2.1 and heterotetrameric Kv2.1/silent Kv6.4 channels.     

Aquaporin Tetramer Composition Modifies the Function of Tobacco Aquaporins

Heterologous expression in yeast cells revealed that NtAQP1, a member of the so-called PIP1 aquaporin subfamily, did not display increased water transport activity in comparison with controls. Instead, an increased CO₂-triggered intracellular acidification was observed. NtPIP2;1, which belongs to the PIP2 subfamily of plant aquaporins, behaved as a true aquaporin but lacked a CO₂-related function. Results from split YFP experiments, protein chromatography, and gel electrophoresis indicated that the proteins form heterotetramers when coexpressed in yeast. Tetramer composition had effects on transport activity as demonstrated by analysis of artificial heterotetramers with a defined proportion of NtAQP1 to NtPIP2;1. A single NtPIP2;1 aquaporin in a tetramer was sufficient to significantly increase the water permeability of the respective yeast cells. With regard to CO₂-triggered intracellular acidification, a cooperative effect was observed, where maximum rates were measured when the tetramer consisted of NtAQP1 aquaporins only. The results confirm the model of an aquaporin monomer as a functional unit for water transport and suggest that, for CO₂-related transport processes, a structure built up by the tetramer is the basis of this function.     

Functional study of a KCNH2 mutant: Novel insights on the pathogenesis of the LQT2 syndrome

The K⁺ voltage‐gated channel subfamily H member 2 (KCNH2) transports the rapid component of the cardiac delayed rectifying K⁺ current. The aim of this study was to characterize the biophysical properties of a C‐terminus‐truncated KCNH2 channel, G1006fs/49 causing long QT syndrome type II in heterozygous members of an Italian family. Mutant carriers underwent clinical workup, including 12‐lead electrocardiogram, transthoracic echocardiography and 24‐hour ECG recording. Electrophysiological experiments compared the biophysical properties of G1006fs/49 with those of KCNH2 both expressed either as homotetramers or as heterotetramers in HEK293 cells. Major findings of this work are as follows: (a) G1006fs/49 is functional at the plasma membrane even when co‐expressed with KCNH2, (b) G1006fs/49 exerts a dominant‐negative effect on KCNH2 conferring specific biophysical properties to the heterotetrameric channel such as a significant delay in the voltage‐sensitive transition to the open state, faster kinetics of both inactivation and recovery from the inactivation and (c) the activation kinetics of the G1006fs/49 heterotetrameric channels is partially restored by a specific KCNH2 activator. The functional characterization of G1006fs/49 homo/heterotetramers provided crucial findings about the pathogenesis of LQTS type II in the mutant carriers, thus providing a new and potential pharmacological strategy.     

Targeted antireceptor therapy with monoclonal antibodies leads to the formation of inactivated tetrameric forms of ErbB receptors

mAbs capable of disabling heterodimeric kinase complexes of the epidermal growth factor receptor (EGFR) and human EGFR type 2/neu have therapeutic relevance to various human cancers. In this study, we demonstrate that in addition to the dimer, EGFR and human EGFR type 2 can associate as homo- and heterotetramers. EGF-induced phosphorylation of the tetramers was significantly lower than that of the dimers, indicating that the tetrameric receptor complexes have impaired signaling activity. Targeting v-erb-b2 erythroblastic leukemia viral oncogene homolog (erbB) receptors with mAbs promoted erbB tetrameric assembly, suggesting that a component of the antitumor activity may be mediated by the ability of Abs to shift the equilibrium from active dimeric to impaired tetrameric receptor complex states. This study suggests a novel therapeutic approach to disable signaling of erbB and potentially other receptors in tumors by biologic agents capable of inducing receptor tetramerization.     

Image processing of electron micrographs of human alpha 2-macroglobulin half-molecules induced by Cd2+

Human alpha 2-macroglobulin is a tetrameric plasma inhibitor of proteinases. Its dissociation by Cd2+ gives functional dimers. Electron microscopy of negatively stained dimers shows their round-ended cylindrical shape with furrows delimiting 3 main stain-excluding domains. Image processing of electron micrographs shows the existence of 2 main orientations of the dimers on the carbon support film. The dimer is composed of 2 curved monomers linked in a central domain, and related by a 90 degree rotation. Taking into account the known primary structure of alpha 2-macroglobulin and the linkage of the 2 constitutive monomers by 2 disulfide bonds, the molecular organization of the dimer is discussed, extended to the tetrameric molecule and compared to the published models of human alpha 2-macroglobulin.     

Characterization of matrix-bound Band 3, the anion transport protein from human erythrocyte membranes

Band 3 (Mr = 95,000), the anion transport protein of human erythrocyte membranes exists primarily as a dimer in solutions of nonionic detergents such as octaethylene glycol mono-n-dodecyl ether (C12E8). The role of the oligomeric structure of Band 3 in the binding of [14C]4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS), an inhibitor of anion transport (Ki = 1-2 microM), was studied by characterizing the interaction of BADS with dimers and monomers of Band 3 covalently attached to p-mercuribenzoate-Sepharose 4B. BADS bound to matrix-bound Band 3 dimers with an affinity of approximately 3 microM at a stoichiometry of 1 BADS molecule/Band 3 monomer, in agreement with the BADS binding characteristic of Band 3 in the membrane and in solutions of C12E8. Band 3 dimers could be attached to the matrix via one subunit by limiting the amount of p-chloromercuribenzoate on the Sepharose bead. Matrix-bound monomers were formed by dissociation of the dimers with dodecyl sulfate or guanidine hydrochloride. Complete removal of the denaturants allowed formation of refolded Band 3 monomers since the matrix-bound subunits could not reassociate. These refolded Band 3 monomers were unable to bind BADS. Release of the monomers from the matrix with 2-mercaptoethanol allowed reformation of dimers with recovery of the BADS binding sites. These results suggest that the dimeric structure of Band 3 is required for BADS binding and that the BADS binding sites may be at the interface between the two halves of the Band 3 dimer.     

Loop A Is Critical for the Functional Interaction of Two Beta vulgaris PIP Aquaporins

Research done in the last years strongly support the hypothesis that PIP aquaporin can form heterooligomeric assemblies, specially combining PIP2 monomers with PIP1 monomers. Nevertheless, the structural elements involved in the ruling of homo versus heterooligomeric organization are not completely elucidated. In this work we unveil some features of monomer-monomer interaction in Beta vulgaris PIP aquaporins. Our results show that while BvPIP2;2 is able to interact with BvPIP1;1, BvPIP2;1 shows no functional interaction. The lack of functional interaction between BvPIP2;1 and BvPIP1;1 was further corroborated by dose-response curves of water permeability due to aquaporin activity exposed to different acidic conditions. We also found that BvPIP2;1 is unable to translocate BvPIP1;1-ECFP from an intracellular position to the plasma membrane when co-expressed, as BvPIP2;2 does. Moreover we postulate that the first extracellular loop (loop A) of BvPIP2;1, could be relevant for the functional interaction with BvPIP1;1. Thus, we investigate BvPIP2;1 loop A at an atomic level by Molecular Dynamics Simulation (MDS) and by direct mutagenesis. We found that, within the tetramer, each loop A presents a dissimilar behavior. Besides, BvPIP2;1 loop A mutants restore functional interaction with BvPIP1;1. This work is a contribution to unravel how PIP2 and PIP1 interact to form functional heterooligomeric assemblies. We postulate that BvPIP2;1 loop A is relevant for the lack of functional interaction with BvPIP1;1 and that the monomer composition of PIP assemblies determines their functional properties.     

The subcellular localization of an aquaporin-2 tetramer depends on the stoichiometry of phosphorylated and nonphosphorylated monomers

In renal principal cells, vasopressin regulates the shuttling of the aquaporin (AQP)2 water channel between intracellular vesicles and the apical plasma membrane. Vasopressin-induced phosphorylation of AQP2 at serine 256 (S256) by protein kinase A (PKA) is essential for its localization in the membrane. However, phosphorylated AQP2 (p-AQP2) has also been detected in intracellular vesicles of noninduced principal cells. As AQP2 is expressed as homotetramers, we hypothesized that the number of p-AQP2 monomers in a tetramer might be critical for the its steady state distribution. Expressed in oocytes, AQP2-S256D and AQP2-S256A mimicked p-AQP2 and non-p-AQP2, respectively, as routing and function of AQP2-S256D and wild-type AQP2 (wt-AQP2) were identical, whereas AQP2-S256A was retained intracellularly. In coinjection experiments, AQP2-S256A and AQP2-S256D formed heterotetramers. Coinjection of different ratios of AQP2-S256A and AQP2-S256D cRNAs revealed that minimally three AQP2-S256D monomers in an AQP2 tetramer were essential for its plasma membrane localization. Therefore, our results suggest that in principal cells, minimally three monomers per AQP2 tetramer have to be phosphorylated for its steady state localization in the apical membrane. As other multisubunit channels are also regulated by phosphorylation, it is anticipated that the stoichiometry of their phosphorylated and nonphosphorylated subunits may fine-tune the activity or subcellular localization of these complexes.     

Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress

Most of the symplastic water transport in plants occurs via aquaporins, but the extent to which aquaporins contribute to plant water status under favorable growth conditions and abiotic stress is not clear. To address this issue, we constitutively overexpressed the Arabidopsis plasma membrane aquaporin, PIP1b, in transgenic tobacco plants. Under favorable growth conditions, PIP1b overexpression significantly increased plant growth rate, transpiration rate, stomatal density, and photosynthetic efficiency. By contrast, PIP1b overexpression had no beneficial effect under salt stress, whereas during drought stress it had a negative effect, causing faster wilting. Our results suggest that symplastic water transport via plasma membrane aquaporins represents a limiting factor for plant growth and vigor under favorable conditions and that even fully irrigated plants face limited water transportation. By contrast, enhanced symplastic water transport via plasma membrane aquaporins may not have any beneficial effect under salt stress, and it has a deleterious effect during drought stress.     

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Phosphatidylinositol bisphosphate (PIP2) is the most abundant phosphoinositide in the plasma membranes of cells and its interaction with many ion channel proteins has proven to be a critical factor enabling ion channel gating. All members of the inwardly rectifying potassium (Kir) channel family depend on PIP2 for their activity, displaying distinct affinities and stereospecificities of interaction with the phosphoinositide. Here, we explored the stoichiometry of Kir channels with PIP2. We first showed that PIP2 regulated the activity of Kir3.4 channels mainly by altering their bursting behavior. Detailed burst analysis indicates that the channels assumed up to four open states and a connectivity of four between open and closed states depending on the available PIP2 levels. Moreover, by controlling the number of PIP2-sensitive subunits in the stoichiometry of a tetrameric Kir2.1 channel, we showed that characteristic channel activity was obtained when at least two wild-type subunits were present. Our studies support a kinetic model for gating of Kir channels by PIP2, where each of the four open states corresponds to the channel activated by one to four PIP2 molecules.     

Methylation of aquaporins in plant plasma membrane

A thorough analysis, using MS, of aquaporins expressed in plant root PM (plasma membrane) was performed, with the objective of revealing novel post-translational regulations. Here we show that the N-terminal tail of PIP (PM intrinsic protein) aquaporins can exhibit multiple modifications and is differentially processed between members of the PIP1 and PIP2 subclasses. Thus the initiating methionine was acetylated or cleaved in native PIP1 and PIP2 isoforms respectively. In addition, several residues were detected to be methylated in PIP2 aquaporins. Lys3 and Glu6 of PIP2;1, one of the most abundant aquaporins in the PM, occurred as di- and mono-methylated residues respectively. Ectopic expression in Arabidopsis suspension cells of PIP2;1, either wild-type or with altered methylation sites, revealed an interplay between methylation at the two sites. Measurements of water transport in PM vesicles purified from these cells suggested that PIP2;1 methylation does not interfere with the aquaporin intrinsic water permeability. In conclusion, the present study identifies methylation as a novel post-translational modification of aquaporins, and even plant membrane proteins, and may represent a critical advance towards the identification of new regulatory mechanisms of membrane transport.     

Water channels in the plant plasma membrane cloned by immunoselection from a mammalian expression system

Expression in mammalian COS cells and an efficient microtiter-based strategy for immunoselection was used in a novel approach to identify genes encoding plant membrane proteins. COS cells were transfected with an Arabidopsis thaliana root cDNA library constructed in a bacterial mammalian shuttle vector and screened with an antiserum raised against purified deglycosylated integral plasma membrane proteins from A. thaliana roots. Antibodies directed against a prominent 27 kDa antigen led to the identification of five different genes. They comprised two subfamilies related to the major intrinsic protein (MIP) superfamily and were named plasma membrane intrinsic proteins, PIP1 and PIP2, since the cellular localization of PIP1 and most probably PIP2 proteins in the plasma membrane was independently confirmed by their co-segregation with marker enzymes during aequeous two-phase partitioning. Surprisingly, expression in Xenopus laevis oocytes revealed that all five PIP mRNAs coded for Hg²⁺-sensitive water transport facilitating activities. There had been no previous evidence of the existence of water channels in the plasma membrane of plant cells and the high diffusional water permeability of the lipid bilayer was considered to be sufficient for water exchange. Nevertheless, Northern and Western analyses showed that the PIP genes are constitutively and possibly even redundantly expressed from the small A. thaliana genome.