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1.
Int J Mol Sci ; 21(22)2020 Nov 10.
Article in English | MEDLINE | ID: mdl-33182845

ABSTRACT

Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49-Y51-Q52) and transmembrane domain 2 (G326-K327-F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel.


Subject(s)
Receptors, Purinergic P2X7/genetics , Receptors, Purinergic P2X7/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Conserved Sequence , HEK293 Cells , Humans , Ion Channel Gating , Kinetics , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Domains , Protein Interaction Domains and Motifs , Rats , Receptors, Purinergic P2X7/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Static Electricity
2.
BMC Mol Biol ; 16: 5, 2015 Mar 11.
Article in English | MEDLINE | ID: mdl-25888347

ABSTRACT

BACKGROUND: With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-amplification reaction and propose the optimal, general setup for gene expression experiment using BioMark instrument (Fluidigm). RESULTS: For evaluating different pre-amplification factors following conditions were combined: four human blood samples from healthy donors and five transcripts having high to low expression levels; each cDNA sample was pre-amplified at four cycles (15, 18, 21, and 24) and five concentrations (equivalent to 0.078 ng, 0.32 ng, 1.25 ng, 5 ng, and 20 ng of total RNA). Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20 ng of total RNA with 15 cycles of pre-amplification, 20x and 40x diluted; and 5 ng and 20 ng of total RNA with 18 cycles of pre-amplification, both 20x and 40x diluted. CONCLUSIONS: We set up upper limits for the bulk gene expression experiment using gene expression Dynamic Array and provided an easy-to-obtain tool for measuring of pre-amplification success. We also showed that variability of the pre-amplification, introduced into the experimental workflow of reverse transcription-qPCR, is lower than variability caused by the reverse transcription step.


Subject(s)
Gene Expression Profiling/instrumentation , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Gene Expression Profiling/methods , Healthy Volunteers , Humans , RNA, Messenger/analysis , RNA, Messenger/blood , Reverse Transcriptase Polymerase Chain Reaction/instrumentation , Sensitivity and Specificity
3.
J Neurochem ; 133(6): 815-27, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25712548

ABSTRACT

In the sustained presence of agonist, the opening of P2X7R channel is followed by pore dilatation, which causes an increase in its permeability to larger organic cations, accompanied by receptor sensitization. To explore the molecular mechanisms by which the conductivity and sensitivity are increased, we analyzed the electrophysiological properties and YO-PRO-1 uptake of selected alanine mutants in the first and second transmembrane domains of the rat P2X7R. Substitution of residues Y40, F43, G338, and D352 with alanine reduced membrane trafficking, and the D352A was practically non-functional. The Y40A and F43A mutants that were expressed in the membrane lacked pore dilation ability. Moreover, the Y40A and Y40F displayed desensitization, whereas the Y40W partially recovered receptor function. The G338A/S mutations favored the open state of the channel and displayed instantaneous permeability to larger organic cations. The G338P was non-functional. The L341A and G345A displayed normal trafficking, current amplitude, and sensitization, but both mutations resulted in a decreased pore formation and dye uptake. These results showed that the increase in P2X7R conductivity and sensitivity is critically dependent on residues Y40 and F43 in the TM1 domain and that the region located at the intersection of TM2 helices controls the rate of large pore opening. We investigated the mechanism of the proapoptotic receptor P2X7R's large pore opening and its sensitization. We found that aromatic residues in the upper part of the first transmembrane domain (TM1) are critical for both the P2X7R channel pore opening and receptor sensitization, and residues located at or below the intersection of the second transmembrane domains (TM2) control the rate of pore opening. These findings identify new residues involved in pore formation of P2X7R.


Subject(s)
Receptors, Purinergic P2X7/chemistry , Receptors, Purinergic P2X7/metabolism , Amino Acid Sequence , Animals , HEK293 Cells , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Patch-Clamp Techniques , Protein Structure, Tertiary , Protein Transport , Rats , Transfection
4.
Mol Pharmacol ; 82(5): 910-7, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22874415

ABSTRACT

Nicotinic acetylcholine receptors (nAChRs) are oligomeric transmembrane proteins in which five subunits coassemble to form a central ion channel pore. Conventional agonists, such as acetylcholine (ACh), bind to an orthosteric site, located at subunit interfaces in the extracellular domain. More recently, it has been demonstrated that nAChRs can also be activated by ligands binding to an allosteric transmembrane site. In the case of α7 nAChRs, ACh causes rapid activation and almost complete desensitization. In contrast, allosteric agonists such as 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quin oline-8-sulfonamide (4BP-TQS) activate α7 nAChRs more slowly and cause only low levels of apparent desensitization. In the present study, single-channel patch-clamp recording has been used to investigate differences in the mechanism of activation of α7 nAChRs by ACh and 4BP-TQS. The most striking difference between activation by ACh and 4BP-TQS is in single-channel kinetics. In comparison with activation by ACh, single-channel open times and burst lengths are substantially longer (~160-800-fold, respectively), and shut times are shorter (~8-fold) when activated by 4BP-TQS. In addition, coapplication of ACh and 4BP-TQS results in a further increase in single-channel burst lengths. Mean burst lengths seen when the two agonists are coapplied (3099 ± 754 ms) are ~2.5-fold longer than with 4BP-TQS alone and ∼370-fold longer than with ACh alone. Intriguingly, the main single-channel conductance of α7 nAChRs, was significantly larger when activated by 4BP-TQS (100.3 ± 2.4 pS) than when activated by ACh (90.0 ± 2.7 pS), providing evidence that activation by allosteric and orthosteric agonists results in different α7 nAChRs open-channel conformations.


Subject(s)
Acetylcholine/pharmacology , Nicotinic Agonists/pharmacology , Quinolines/pharmacology , Receptors, Nicotinic/metabolism , Sulfonamides/pharmacology , Allosteric Regulation , Animals , Drug Interactions , Female , Humans , Ion Channel Gating , Kinetics , Oocytes/drug effects , Oocytes/physiology , Patch-Clamp Techniques , Xenopus laevis , alpha7 Nicotinic Acetylcholine Receptor
5.
PLoS One ; 7(2): e32073, 2012.
Article in English | MEDLINE | ID: mdl-22359659

ABSTRACT

We have examined the effect of temperature on the electrophysiological properties of three neuronal nicotinic acetylcholine receptor (NACHR) subtypes: the rapidly desensitizing homomeric α7 nAChR, the more slowly desensitizing heteromeric α4ß2 nAChR and on α7 nAChRs containing a transmembrane mutation (L247T) that results in dramatically reduced desensitization. In all cases, the functional properties of receptors expressed in Xenopus oocytes at room temperature (RT; 21°C) were compared to those recorded at either physiological temperature (37°C) or at lower temperature (4°C). Alterations in temperature had dramatically differing effects on the amplitude of whole-cell responses detected with these three nAChR subtypes. Compared to responses at RT, the amplitude of agonist-evoked responses with α4ß2 nAChRs was increased at high temperature (125±9%, n = 6, P<0.01) and reduced at low temperature (47±5%, n = 6, P<0.01), whereas the amplitude of α7 responses was reduced at high temperature (27±7%, n = 11, P<0.001) and increased at low temperatures (224±16%, n = 10, P<0.001). In contrast to the effects of temperature on α4ß2 and wild type α7 nAChRs, the amplitude of α7 nAChRs containing the L247T mutation was unaffected by changes in temperature. In addition, changes in temperature had little or no effect on current amplitude when α7 nAChRs were activated by the largely non-desensitizing allosteric agonist 4BP-TQS. Despite these differing effects of temperature on the amplitude of agonist-evoked responses in different nAChRs, changes in temperature had a consistent effect on the rate of receptor desensitization on all subtypes examined. In all cases, higher temperature resulted in increased rates of desensitization. Thus, it appears that the differing effects of temperature on the amplitudes of whole-cell responses cannot be explained by temperature-induced changes in receptor desensitization rates.


Subject(s)
Electrophysiological Phenomena , Receptors, Nicotinic/physiology , Temperature , Animals , Electrophysiological Phenomena/drug effects , Humans , Patch-Clamp Techniques , Transduction, Genetic , Xenopus laevis , alpha7 Nicotinic Acetylcholine Receptor
6.
Purinergic Signal ; 8(2): 317-25, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22286664

ABSTRACT

The P2X7 receptor (P2X7R) is a member of the ATP-gated ion channel family that exhibits distinct electrophysiological and pharmacological properties. This includes low sensitivity to ATP, lack of desensitization, a sustained current growth during prolonged receptor stimulation accompanied with development of permeability to large organic cations, and the coupling of receptor activation to cell blebbing and death. The uniquely long C-terminus of P2X7R accounts for many of these receptor-specific functions. The aim of this study was to understand the role of conserved ectodomain cysteine residues in P2X7R function. Single- and double-point threonine mutants of C119-C168, C129-C152, C135-C162, C216-C226, and C260-C269 cysteine pairs were expressed in HEK293 cells and studied using whole-cell current recording. All mutants other than C119T-P2X7R responded to initial and subsequent application of 300-µM BzATP and ATP with small amplitude monophasic currents or were practically nonfunctional. The mutagenesis-induced loss of function was due to decreased cell-surface receptor expression, as revealed by assessing levels of biotinylated mutants. Coexpression of all double mutants with the wild-type receptor had a transient or, in the case of C119T/C168T double mutant, sustained inhibitory effect on receptor trafficking. The C119T-P2X7R mutant was expressed on the plasma membrane and was fully functional with a slight decrease in the sensitivity for BzATP, indicating that interaction of liberated Cys168 with another residue rescues the trafficking of receptor. Thus, in contrast to other P2XRs, all disulfide bonds of P2X7R are individually essential for the proper receptor trafficking.


Subject(s)
Conserved Sequence , Cysteine/physiology , Receptors, Purinergic P2X7/metabolism , Animals , Cysteine/biosynthesis , Cysteine/genetics , HEK293 Cells , Humans , Mutation/physiology , Protein Transport/physiology , Rats , Receptors, Purinergic P2X7/genetics
7.
J Neurochem ; 119(4): 676-85, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21883226

ABSTRACT

Tyrosine 37 in the first transmembrane (TM1) domain is highly conserved in ATP-gated P2X receptors suggesting its fundamental role. We tested whether Y37 contributes to the desensitization of P2X3 receptors, which is currently not well understood. By combining electrophysiological, imaging and modeling approaches, we studied desensitization of various Y37 P2X3 mutants and potential partners of Y37. Unlike the membrane current of the WT receptor, which desensitized in seconds, Y37A mutant current did not fully desensitize even after minutes-long applications of ß,γ-meATP, α,ß-meATP, ATP or 2MeS-ATP. The fractional calcium current was enhanced in the Y37A mutant. Y37F did not rescue the native P2X3 phenotype indicating a role for the hydroxyl group of Y37 for the WT receptor. Homology modeling indicated I318 or I319 in TM2 as potential partners for Y37 in the receptor closed state. We tested this hypothesis by creating a permanent interaction between the two residues via disulfide bond. Whereas single Y37C, I318C and I319C mutants were functional, the double mutants Y37C-I318C and Y37C-I319C were non-functional. Using a cyclic model of receptor operation, we suggest that the conserved tyrosine 37 links TM1 to TM2 of adjacent subunit to stabilize desensitized states and restricts calcium permeability through the ion channel.


Subject(s)
Adenosine Triphosphate/metabolism , Calcium/metabolism , Receptors, Purinergic P2X3/metabolism , Tyrosine/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Alanine/genetics , Animals , Calcium Signaling/drug effects , Calcium Signaling/genetics , Cell Line, Transformed , Computer Simulation , Cysteine/genetics , Dose-Response Relationship, Drug , Green Fluorescent Proteins/genetics , Humans , Ion Channel Gating/drug effects , Ion Channel Gating/genetics , Membrane Potentials/drug effects , Membrane Potentials/genetics , Models, Molecular , Mutation/genetics , Patch-Clamp Techniques , Permeability , Rats , Receptors, Purinergic P2X3/genetics , Silver Nitrate/pharmacology , Transfection/methods , Tyrosine/genetics
8.
J Neurochem ; 109(3): 923-34, 2009 May.
Article in English | MEDLINE | ID: mdl-19425179

ABSTRACT

The functional relevance of aromatic residues in the upper part of the transmembrane domain-1 of purinergic P2X receptors (P2XRs) was examined. Replacement of the conserved Tyr residue with Ala had a receptor-specific effect: the P2X1R was non-functional, the P2X2R, P2X4R, and P2X3R exhibited enhanced sensitivity to ATP and alphabeta-meATP accompanied by prolonged decay of current after washout of agonists, and the P2X7R sensitivity for agonists was not affected, though decay of current was delayed. The replacement of the P2X4R-Tyr42 with other amino acids revealed the relevance of an aromatic residue at this position. Mutation of the neighboring Phe and ipsilateral Tyr/Trp residues, but not the contralateral Phe residue, also affected the P2X2R, P2X3R, and P2X4R function. Double mutation of ipsilateral Tyr42 and Trp46 P2X4R residues restored receptor function, whereas the corresponding P2X2R double mutant was not functional. In contrast, mutation of the contralateral Phe48 residue in the P2X4R-Y42A mutant had no effect. These results indicate that aromatic residues in the upper part of TM1 play important roles in the three-dimensional structure of the P2XRs and that they are required not only for ion conductivity but also for specificity of agonist binding and/or channel gating.


Subject(s)
Amino Acids, Aromatic/metabolism , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Amino Acid Sequence , Amino Acids, Aromatic/genetics , Biophysics , Cell Line, Transformed , Dose-Response Relationship, Drug , Electric Stimulation , Green Fluorescent Proteins/genetics , Humans , Membrane Potentials/drug effects , Membrane Potentials/genetics , Mutagenesis/genetics , Patch-Clamp Techniques/methods , Protein Binding/genetics , Protein Binding/physiology , Protein Conformation , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , Receptors, Purinergic P2/classification , Signal Transduction/drug effects , Signal Transduction/physiology , Transfection/methods
9.
Pflugers Arch ; 456(5): 939-50, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18427835

ABSTRACT

Ivermectin (IVM), a large macrocyclic lactone, specifically enhances P2X(4) receptor-channel function by interacting with residues of transmembrane (TM) helices in the open conformation state. In this paper, we used cysteine-scanning mutagenesis of rat P2X(4)-TMs to identify and map residues of potential importance for channel gating and interaction with IVM. The receptor function was unchanged by mutations in 29 different residues, and among them, the IVM effects were altered in Gln(36), Leu(40), Val(43), Val(47), Trp(50), Asn(338), Gly(342), Leu(346), Ala(349), and Ile(356) mutants. The substitution-sensitive Arg(33) and Cys(353) mutants could also be considered as IVM-sensitive hits. The pattern of these 12 residues was consistent with helical topology of both TMs, with every third or fourth amino acid affected by substitution. These predominantly hydrophobic-nonpolar residues are also present in the IVM-sensitive Schistosoma mansoni P2X subunit. They lie on the same side of their helices and could face lipids in the open conformation state and provide the binding pocket for IVM. In contrast, the IVM-independent hits Met(31), Tyr(42), Gly(45), Val(49), Gly(340), Leu(343), Ala(344), Gly(347), Thr(350), Asp(354), and Val(357) map on the opposite side of their helices, probably facing the pore of receptor or protein and playing important roles in gating.


Subject(s)
Antiparasitic Agents/metabolism , Ion Channel Gating , Ivermectin/metabolism , Protein Conformation , Receptors, Purinergic P2/chemistry , Receptors, Purinergic P2/metabolism , Amino Acid Sequence , Animals , Cell Line , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Patch-Clamp Techniques , Rats , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2X4
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