Alternative Splicing of the N-Terminal Cytosolic and Transmembrane Domains of P2X7 Controls Gating of the Ion Channel by ADP-Ribosylation

P2X7 is a homotrimeric ion channel with two transmembrane domains and a large extracellular ATP-binding domain. It plays a key role in the response of immune cells to danger signals released from cells at sites of inflammation. Gating of murine P2X7 can be induced by the soluble ligand ATP, as well as by NAD(+)-dependent ADP-ribosylation of arginine 125, a posttranslational protein modification catalyzed by the toxin-related ecto-enzymes ART2.1 and ART2.2. R125 is located at the edge of the ligand-binding crevice. Recently, an alternative splice variant of P2X7, designated P2X7(k), was discovered that differs from the previously described variant P2X7(a) in the N-terminal 42 amino acid residues composing the first cytosolic domain and most of the Tm1 domain. Here we compare the two splice variants of murine P2X7 with respect to their sensitivities to gating by ADP-ribosylation in transfected HEK cells. Our results show that the P2X7(k) variant is sensitive to activation by ADP-ribosylation whereas the P2X7(a) variant is insensitive, despite higher cell surface expression levels. Interestingly, a single point mutation (R276K) renders the P2X7(a) variant sensitive to activation by ADP-ribosylation. Residue 276 is located at the interface of neighboring subunits approximately halfway between the ADP-ribosylation site and the transmembrane domains. Moreover, we show that naive and regulatory T cells preferentially express the more sensitive P2X7(k) variant, while macrophages preferentially express the P2X7(a) variant. Our results indicate that differential splicing of alternative exons encoding the N-terminal cytosolic and transmembrane domains of P2X7 control the sensitivity of different immune cells to extracellular NAD(+) and ATP.     

Identification of a domain involved in ATP-gated ionotropic receptor subunit assembly.

P2X receptors are ATP-gated ion channels found in a variety of tissues and cell types. Seven different subunits (P2X(1)-P2X(7)) have been molecularly cloned and are known to form homomeric, and in some cases heteromeric, channel complexes. However, the molecular determinants leading to the assembly of subunits into P2X receptors are unknown. To address this question we utilized a co-immunoprecipitation assay in which epitope-tagged deletion mutants and chimeric constructs were examined for their ability to co-associate with full-length P2X subunits. Deletion mutants of the P2X(2) receptor subunit were expressed individually and together with P2X(2) or P2X(3) receptor subunits in HEK 293 cells. Deletion of the amino terminus up to the first transmembrane domain (amino acid 28) and beyond (to amino acid 51) did not prevent subunit assembly. Analysis of the carboxyl terminus demonstrated that mutants missing the portion of the protein downstream of the second transmembrane domain could also still co-assemble. However, a mutant terminating 25 amino acids before the second transmembrane domain could not assemble with other subunits or itself, implicating the missing region of the protein in assembly. This finding was supported and extended by data utilizing a chimera strategy that indicated TMD2 is a critical determinant of P2X subunit assembly.     

The first transmembrane domain of the P2X receptor subunit participates in the agonist-induced gating of the channel.

Based on pharmacological properties, the P2X receptor family can be subdivided into those homo-oligomers that are sensitive to the ATP analog alphabeta-methylene ATP(alphabetameATP) (P2X(1) and P2X(3)) and those that are not (P2X(2), P2X(4), P2X(5), P2X(6), and P2X(7)). We exploited this dichotomy through the construction of chimeric receptors and site-directed mutagenesis in order to identify domains responsible for these differences in the abilities of extracellular agonists to gate P2X receptors. Replacement of the extracellular domain of the alphabetameATP-sensitive rat P2X(1) subunit with that of the alphabetameATP-insensitive rat P2X(2) subunit resulted in a receptor that was still alphabetameATP-sensitive, suggesting a non-extracellular domain was responsible for the differential gating of P2X receptors by various agonists. Replacement of the first transmembrane domain of the rat P2X(2) subunit with one from an alphabetameATP-sensitive subunit (either rat P2X(1) or P2X(3) subunit) converted the resulting chimera to alphabetameATP sensitivity. This conversion did not occur when the first transmembrane domain came from a non-alphabetameATP-sensitive subunit. Site-directed mutagenesis indicated that the C-terminal portion of the first transmembrane domain was important in determining the agonist selectivity of channel gating for these chimeras. These results suggest that the first transmembrane domain plays an important role in the agonist operation of the P2X receptor.     

The transmembrane domain of murine alpha-mannosidase IB is a major determinant of Golgi localization

Murine alpha1,2-mannosidase IB is a type II transmembrane protein localized to the Golgi apparatus where it is involved in the biogenesis of complex and hybrid N-glycans. This enzyme consists of a cytoplasmic tail, a transmembrane domain followed by a "stem" region and a large C-terminal catalytic domain. To analyze the determinants of targeting, we constructed various deletion mutants of murine alpha1,2-mannosidase IB as well as alpha1,2-mannosidase IB/yeast alpha1,2-mannosidase and alpha1,2-mannosidase IB/GFP chimeras and localized these proteins by fluorescence microscopy, when expressed transiently in COS7 cells. Replacing the catalytic domain of alpha1,2-mannosidase IB with that of the homologous yeast alpha1,2-mannosidase and deleting the "stem" region in this chimera had no effect on Golgi targeting, but caused increased cell surface localization. The N-terminal tagged protein lacking a catalytic domain was also localized to the Golgi. In the latter case, when the stem region was partially or completely removed, the protein was found in both the ER and the Golgi. A chimera consisting of the alpha1,2-mannosidase IB N-terminal region (cytoplasmic and transmembrane domains plus 10 amino acids of the "stem" region) and GFP was localized mainly to the Golgi. Deletion of 30 out of 35 amino acids in the cytoplasmic tail had no effect on Golgi localization. A GFP chimera lacking the entire cytoplasmic tail was found in both the ER and the Golgi. These results indicate that the transmembrane domain of alpha1,2-mannosidase IB is a major determinant of Golgi localization.     

The P2X7 carboxyl tail is a regulatory module of P2X7 receptor channel activity

P2X(7) receptors are ATP-gated cation channels composed of three identical subunits, each having intracellular amino and carboxyl termini and two transmembrane segments connected by a large ectodomain. Within the P2X family, P2X(7) subunits are unique in possessing an extended carboxyl tail. We expressed the human P2X(7) subunit as two complementary fragments, a carboxyl tail-truncated receptor channel core (residues 1-436 or 1-505) and a tail extension (residues 434-595) in Xenopus laevis oocytes. P2X(7) channel core subunits efficiently assembled as homotrimers that appeared abundantly at the oocyte surface, yet produced only approximately 5% of the full-length P2X(7) receptor current. Co-assembly of channel core subunits with full-length P2X(7) subunits inhibited channel current, indicating that the lack of a single carboxyl tail domain is dominant-negative for P2X(7) receptor activity. Co-expression of the tail extension as a discrete protein increased ATP-gated current amplitudes of P2X(7) channel cores 10-20-fold, fully reconstituting the wild type electrophysiological phenotype of the P2X(7) receptor. Chemical cross-linking revealed that the discrete tail extension bound with unity stoichiometry to the carboxyl tail of the P2X(7) channel core. We conclude that a non-covalent association of crucial functional importance exists between the carboxyl tail of the channel core and the tail extension. Using a slightly shorter P2X(7) subunit core and subfragments of the tail extension, this association could be narrowed down to include residues 409-436 and 434-494 of the split receptor. Together, these results identify the tail extension as a regulatory gating module, potentially making P2X(7) channel gating sensitive to intracellular regulation.     

On the role of the first transmembrane domain in cation permeability and flux of the ATP-gated P2X2 receptor

P2X receptors are a family of seven ligand-gated ion channels (P2X1-P2X7) that open in the presence of ATP. We used alanine-scanning mutagenesis and patch clamp photometry to study the role of the first transmembrane domain of the rat P2X2 receptor in cation permeability and flux. Three alanine-substituted mutants did not respond to ATP, and 19 of the 22 functional receptors resembled the wild-type receptor with regard to the fraction of the total ATP-gated current carried by calcium or the permeability of calcium relative to cesium. The remaining three mutants showed modest changes in calcium dynamics. Two of these occurred at sites (Gly30 and Phe44) that are unlikely to interact with permeating cations in a meaningful way. The third was a conserved tyrosine (Tyr43) that may form an inter-pore binding site for calcium. The data suggest that, with the possible exception of Tyr43, the first transmembrane domain contributes little to the permeation properties of the P2X2 receptor.     

A natural dominant negative P2X1 receptor due to deletion of a single amino acid residue

The P2X1 receptor belongs to a family of oligomeric ATP-gated ion channels with intracellular N and C termini and two transmembrane segments separating a large extracellular domain. Here, we describe a naturally occurring dominant negative P2X1 mutant. This mutant lacks one leucine within a stretch of four leucine residues in its second transmembrane domain (TM2) (amino acids 351-354). Confocal microscopy revealed proper plasma membrane localization of the mutant in stably transfected HEK293 cells. Nevertheless, voltage-clamped HEK293 cells expressing mutated P2X1 channels failed to develop an ATP or ADP-induced current. Furthermore, when co-expressed with the wild type receptor in Xenopus oocytes, the mutated protein exhibited a dose-dependent dominant negative effect on the normal ATP or ADP-induced P2X1 channel activity. These data indicate that deletion of a single apolar amino acid residue at the inner border of the P2X1 TM2 generates a nonfunctional channel. The inactive and dominant negative form of the P2X1 receptor may constitute a new tool for the study of the physiological role of this channel in native cells.     

Effect of cytoplasmic tail truncations on the activity of the M(2) ion channel of influenza A virus

The M(2) protein of influenza A virus forms a proton channel that is required for viral replication. The M(2) ion channel is a homotetramer and has a 24-residue N-terminal extracellular domain, a 19-residue transmembrane domain, and a 54-residue cytoplasmic tail. We show here that the N-terminal methionine residue is cleaved from the mature protein. Translational stop codons were introduced into the M(2) cDNA at residues 46, 52, 62, 72, 77, 82, 87, and 92. The deletion mutants were designated truncx, according to the amino acid position that was changed to a stop codon. We studied the role of the cytoplasmic tail by measuring the ion channel activity (the current sensitive to the M(2)-specific inhibitor amantadine) of the cytoplasmic tail truncation mutants expressed in oocytes of Xenopus laevis. When their conductance was measured over time, mutants trunc72, trunc77, and trunc92 behaved comparably to wild-type M(2) protein (a decrease of only 4% over 30 min). In contrast, conductance decreased by 28% for trunc82, 27% for trunc62, and 81% for trunc52 channels. Complete closure of the channel could be observed in some cells for trunc62 and trunc52 within 30 min. These data suggest that a role of the cytoplasmic tail region of the M(2) ion channel is to stabilize the pore against premature closure while the ectodomain is exposed to low pH.     

Influenza A virus can undergo multiple cycles of replication without M2 ion channel activity

Ion channel proteins are common constituents of cells and have even been identified in some viruses. For example, the M2 protein of influenza A virus has proton ion channel activity that is thought to play an important role in viral replication. Because direct support for this function is lacking, we attempted to generate viruses with defective M2 ion channel activity. Unexpectedly, mutants with apparent loss of M2 ion channel activity by an in vitro assay replicated as efficiently as the wild-type virus in cell culture. We also generated a chimeric mutant containing an M2 protein whose transmembrane domain was replaced with that from the hemagglutinin glycoprotein. This virus replicated reasonably well in cell culture but showed no growth in mice. Finally, a mutant lacking both the transmembrane and cytoplasmic domains of M2 protein grew poorly in cell culture and showed no growth in mice. Thus, influenza A virus can undergo multiple cycles of replication without the M2 transmembrane domain responsible for ion channel activity, although this activity promotes efficient viral replication.     

Identification of the phosphorylation sites on intact TRPM7 channels from mammalian cells

Transient receptor potential melastatin 7 (TRPM7) channels are divalent cation-selective ion channels that are permeable to Ca(2+) and Mg(2+). TRPM7 is ubiquitously expressed in vertebrate cells and contains both an ion channel and a kinase domain. TRPM7 plays an important role in regulating cellular homeostatic levels of Ca(2+) and Mg(2+) in mammalian cells. Although studies have shown that the kinase domain of TRPM7 is required for channel activation and can phosphorylate other target proteins, a systematic analysis of intact TRPM7 channel phosphorylation sites expressed in mammalian cells is lacking. We applied mass spectrometric proteomic techniques to identify and characterize the key phosphorylation sites in TRPM7 channels. We identified 14 phosphorylation sites in the cytoplasmic domain of TRPM7, eight of which have not been previously reported. The identification of phosphorylation sites using antibody-based immunopurification and mass spectrometry is an effective approach for defining the phosphorylation status of TRPM7 channels. The present results show that TRPM7 channels are phosphorylated at multiple sites, which serves as a mechanism to modulate the dynamic functions of TRPM7 channels in mammalian cells.     

The calcium-binding EF-hand in polycystin-L is not a domain for channel activation and ensuing inactivation

Polycystin-L (PCL) shares high homology with polycystin-2, the product of polycystic kidney disease gene-2. It was previously shown that the PCL forms a non-selective cation channel activated by calcium influx. However, it remains unclear whether calcium activates/inactivates PCL by binding to the EF-hand motif located on the cytoplasmic carboxyl-terminus. Here we obtained two PCL splice variants from liver (PCL-LV, lacking the EF-hand) and testis (PCL-TS, lacking 45 amino acids on the carboxyl tail) using PCR-based approaches. When expressed in Xenopus oocytes and studied using electrophysiology both splice variants exhibited basal cation channel activity and calcium-induced channel activation. While PCL-TS displayed similar activation to PCL, PCL-LV exhibited a three-fold increased activation. All five PCL C-terminal artificial truncation mutants also exhibited basal and calcium-activated channel activities, in particular the mutant T622X lacking the EF-hand was associated with increased activation. Our data demonstrate that the EF-hand and other parts of the carboxyl tail of PCL are not determinants of channel activation/inactivation although the EF-hand seems to be involved in the modulation of these processes.     

Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes

K_V10.1 is a voltage-gated potassium channel expressed selectively in the mammalian brain but also aberrantly in cancer cells. In this study we identified short splice variants of K_V10.1 resulting from exon-skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lacked the transmembrane domains of the channel and produced cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitated with the full-length channel and induced a robust down-regulation of K_V10.1 current when co-expressed with the full-length form, but their effect was mechanistically different. E65 required a tetramerization domain and induced a reduction in the overall expression of full-length K_V10.1, whereas E70 mainly affected its glycosylation pattern. E65 triggered the activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of noncanonical functions for the oncogenicity of K_V10.1, which need to be considered when ion channels are targeted for cancer therapy.     

Identification of amino acid residues contributing to the pore of a P2X receptor.

P2X receptors are ion channels opened by extracellular ATP. The seven subunits currently known are encoded by different genes. It is thought that each subunit has two transmembrane domains, a large extracellular loop, and intracellular N- and C-termini, a topology which is fundamentally different from that of other ligand-gated channels such as nicotinic acetylcholine or glutamate receptors. We used the substituted cysteine accessibility method to identify parts of the molecule that form the ionic pore of the P2X2 receptor. Amino acids preceding and throughout the second hydrophobic domain (316-354) were mutated individually to cysteine, and the DNAs were expressed in HEK293 cells. For three of the 38 residues (I328C, N333C, T336C), currents evoked by ATP were inhibited by extracellular application of methanethiosulfonates of either charge (ethyltrimethylammonium, ethylsulfonate) suggesting that they lie in the outer vestibule of the pore. For two further substitutions (L338C, D349C) only the smaller ethylamine derivative inhibited the current. L338C was accessible to cysteine modification whether or not the channel was opened by ATP, but D349C was inhibited only when ATP was concurrently applied. The results indicate that part of the pore of the P2X receptor is formed by the second hydrophobic domain, and that L338 and D349 are on either side of the channel 'gate'.     

Isoform specificity of P2X2 purinergic receptor C-terminus binding to tubulin

Adenosine triphosphate (ATP) and other nucleotides can be released in the central and peripheral nervous systems and act as neurotransmitters/neuromodulators. They can activate G-protein coupled receptors and ligand-gated ion channels, which are present throughout the central nervous system (CNS). P2X2 is one of seven known ion channels gated by ATP, and is characterized by having two transmembrane domains, a large extracellular loop and intracellular N- and C-termini. Recently, work from several laboratories has shown that neurotransmitter receptors can interact with other proteins thereby changing their functional attributes. More specifically, it was demonstrated that P2X2 binds beta-tubulin. Our goal was to investigate this interaction, by comparing P2X2 with a naturally occurring splicing variant named P2X2b. These isoforms differ in their C-terminal regions which contain the proposed beta-tubulin-binding domain. Indeed we were able to demonstrate that only the long variant P2X2 binds beta-tubulin I in various biochemical assays. In addition, this interaction can be direct since it also occurred when the P2X2 C-terminus was exposed to purified brain tubulin. When expressed in heterologous cells, P2X2 interacted with beta-tubulin I while present on the outer membrane, as demonstrated by biotinylation of surface proteins. Therefore, the present data strongly support a functional interaction between an ATP-gated channel and the cytoskeleton. Moreover, we show a biochemical difference between the splicing alternatives that might account for novel distinct functional roles.     

Targeting of a lysosomal membrane protein: a tyrosine-containing endocytosis signal in the cytoplasmic tail of lysosomal acid phosphatase is necessary and sufficient for targeting to lysosomes.

Lysosomal acid phosphatase (LAP) is synthesized as a transmembrane protein with a short carboxy-terminal cytoplasmic tail of 19 amino acids, and processed to a soluble protein after transport to lysosomes. Deletion of the membrane spanning domain and the cytoplasmic tail converts LAP to a secretory protein, while deletion of the cytoplasmic tail as well as substitution of tyrosine 413 within the cytoplasmic tail against phenylalanine causes accumulation at the cell surface. A chimeric polypeptide, in which the cytoplasmic tail of LAP was fused to the ectoplasmic and transmembrane domain of hemagglutinin is rapidly internalized and tyrosine 413 of the LAP tail is essential for internalization of the fusion protein. A chimeric polypeptide, in which the membrane spanning domain and cytoplasmic tail of LAP are fused to the ectoplasmic domain of the Mr 46 kd mannose 6-phosphate receptor, is rapidly transported to lysosomes, whereas wild type receptor is not transported to lysosomes. We conclude that a tyrosine containing endocytosis signal in the cytoplasmic tail of LAP is necessary and sufficient for targeting to lysosomes.     

Assembly of gap junction channels: mechanism, effects of calmodulin antagonists and identification of connexin oligomerization determinants.

The assembly of connexins (Cxs) into gap junction intercellular communication channels was studied. An in vitro cell-free synthesis system showed that formation of the hexameric connexon hemichannels involved dimeric and tetrameric connexin intermediates. Cx32 contains two putative cytoplasmic calmodulin-binding sites, and their role in gap junction channel assembly was investigated. The oligomerization of Cx32 into connexons was reversibly inhibited by a calmodulin-binding synthetic peptide, and by W7, a naphthalene sulfonamide calmodulin antagonist. Removing the calmodulin-binding site located at the carboxyl tail of Cx32 limited connexon formation and resulted in an accumulation of intermediate connexin oligomers. This truncation mutant, Cx32Delta215, when transiently expressed in COS-7 cells, accumulated intracellularly and had failed to target to gap junctions. Immunoprecipitation studies suggested that a C-terminal sequence of Cx32 incorporating the calmodulin-binding site was required for the formation of hetero-oligomers of Cx26 and Cx32 but not for Cx32 homomeric association. A chimera, Cx32TM3CFTR, in which the third transmembrane and proposed channel lining sequence of Cx32 was substituted by a transmembrane sequence of the cystic fibrosis transmembrane conductance regulator, did not oligomerize in vitro and it accumulated intracellularly when expressed in COS-7 cells. The results indicate that amino-acid sequences in the third transmembrane domain and a calmodulin-binding domain in the cytoplasmic tail of Cx32 are likely candidates for regulating connexin oligomerization.     

Analysis of the signals for polarized transport of influenza virus (A/WSN/33) neuraminidase and human transferrin receptor, type II transmembrane proteins.

In polarized MDCK cells influenza virus (A/WSN/33) neuraminidase (NA) and human transferrin receptor (TR), type II glycoproteins, when expressed from cloned cDNAs, were transported and accumulated preferentially on the apical and basolateral surfaces, respectively. We have investigated the signals for polarized sorting by constructing chimeras between NA and TR and by making deletion mutants. NATR delta 90, which contains the cytoplasmic tail and transmembrane domain of NA and the ectodomain of TR, was found to be localized predominantly on the apical membrane, whereas TRNA delta 35, containing the cytoplasmic and transmembrane domains of TR and the ectodomain of NA, was expressed preferentially on the basolateral membrane. TR delta 57, a TR deletion mutant lacking 57 amino acids in the TR cytoplasmic tail, did not exhibit any polarized expression and was present on both apical and basolateral surfaces, whereas a deletion mutant (NA delta 28-35) lacking amino acid residues from 28 to 35 in the transmembrane domain of NA resulted in secretion of the NA ectodomain predominantly from the apical side. These results taken together indicate that the cytoplasmic tail of TR was sufficient for basolateral transport, but influenza virus NA possesses two sorting signals, one in the cytoplasmic or transmembrane domain and the other within the ectodomain, both of which are independently able to transport the protein to the apical plasma membrane.     

Molecular structure of purinergic P2X receptors and their expression in the hypothalamus and pituitary

Purinergic P2X receptors represent a novel structural type of ligand-gated ion channels activated by extracellular ATP. So far, seven P2X receptor subunits have been found in excitable as well as non-excitable tissues. Little is known about their structure, mechanism of channel opening, localization, and role in the central nervous system. The aim of this work is to summarize recent investigations and describe our contribution to elucidating the structure of the ATP binding site and transmembrane domains of the P2X receptor, we also discuss the expression and physiological roles played by the ATP and P2X receptors in the anterior pituitary and hypothalamus.     

The role of extracellular and cytoplasmic splice domains of alpha7-integrin in cell adhesion and migration on laminins

The major laminin-binding integrin of skeletal, smooth, and heart muscle is alpha7beta1-integrin, which is structurally related to alpha6beta1. It occurs in three cytoplasmic splice variants (alpha7A, -B, and -C) and two extracellular forms (X1 and X2) which are developmentally regulated and differentially expressed in skeletal muscle. Previously, we have shown that ectopic expression of the alpha7beta-integrin splice variant in nonmotile HEK293 cells specifically induced cell locomotion on laminin-1 but not on fibronectin. To investigate the specificity and the mechanism of the alpha7-mediated cell motility, we expressed the three alpha7-chain cytoplasmic splice variants, as well as alpha6A- and alpha6B-integrin subunits in HEK293 cells. Here we show that all three alpha7 splice variants (containing the X2 domain), as well as alpha6A and alpha6B, promote cell attachment and stimulate cell motility on laminin-1 and its E8 fragment. Deletion of the cytoplasmic domain (excluding the GFFKR consensus sequence) from alpha7B resulted in a loss of the motility-enhancing effect. On laminin-2/4 (merosin), the predominant isoform in mature skeletal muscle, only alpha7-expressing cells showed enhanced motility, whereas cells transfected with alpha6A and alpha6B neither attached nor migrated on laminin-2. Adhesion of alpha7-expressing cells to both laminin-1 and laminin-2 was specifically inhibited by a new monoclonal antibody (6A11) specific for alpha7. Expression of the two extracellular splice variants alpha7X1 and alpha7X2 in HEK293 cells conferred different motilities on laminin isoforms: Whereas alpha7X2B promoted cell migration on both laminin-1 and laminin-2, alpha7X1B supported motility only on laminin-2 and not on laminin-1, although both X1 and X2 splice variants revealed similar adhesion rates to laminin-1 and -2. Fluorescence-activated cell sorter analysis revealed a dramatic reduction of surface expression of alpha6-integrin subunits after alpha7A or -B transfection; also, surface expression of alpha1-, alpha3-, and alpha5-integrins was significantly reduced. These results demonstrate selective responses of alpha6- and alpha7-integrins and of the alpha7 splice variants to laminin-1 and -2 and indicate differential roles in laminin-controlled cell adhesion and migration.