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Cloning and characterization of the ionotropic GABA receptor subunit ρ1 from pig (Sus scrofa)
Neuroscience Letters 558 (2014) 78–81
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Cloning and characterization of the ionotropic GABA receptor subunit 1 from pig (Sus scrofa) Jorge Mauricio Reyes-Ruiz ∗ , Agenor Limon, Ricardo Miledi Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA 92697, USA
h i g h l i g h t s • • • • •
Oocytes injected with retina mRNA generated slow desensitizing GABA currents. A cDNA clone was obtained by RT-PCR and cRNA generated from it. Xoocytes injected with cRNA were used for pharmacological characterization. The cloned nucleotidic sequence is similar to the human transcription variant 2. A 2nd splicing variant was cloned with a stop codon early in the reading frame.
a r t i c l e
i n f o
Article history: Received 5 September 2013 Received in revised form 28 October 2013 Accepted 3 November 2013 Keywords: Xenopus oocyte Electrophysiology mRNA Sequencing ESTs
a b s t r a c t Since human and pig eyes have remarkably anatomical and physiological similitudes swine models have been broadly used for functional studies and therapeutic research. Recently, a GABA-mediated relaxation of retinal vascularity suggested that GABA signaling may be used to improve retinal blood flow in vascular-driven impaired vision, and a further molecular characterization of GABA receptors would be beneficial. However, none of the GABA type subunits from pigs has been yet cloned; Among the 19 subunits that compose the family of GABAA receptors, 1–3 subunits are capable of forming homomeric channels. These homomeric receptors are particularly interesting because their pharmacological and kinetic properties are notably different from receptors composed by other GABAA subunits. Here we report the cloning of the GABA1subunit from the pig and the functional expression of homomeric channels in Xenopus oocytes. The most notable difference found in the pig GABA1 receptor was the absence of a stretch of 17 amino acids near the amino terminus (R41–V58) conserved in the rat and the human. This sequence has a higher nucleotidic match with the transcript variant 2 of the human GABA1 subunit. Xenopus oocytes injected with cRNA from the receptor generated currents when exposed to GABA that shared all the characteristics of other GABA1 subunits in mammals, including its modulation by dopamine. This study will help to increase the knowledge of the genetics of the pig, further the understanding of this important neurotransmitter receptor family and will shed some light in the evolution of these genes among mammals. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Human and pig eyes have remarkably anatomical and physiological similitudes [7,8]; therefore, swine models are preferable to murine models for ontogeny studies and therapeutic research. Recently it was shown that ␥-aminobutyrate (GABA) participates in the relaxation of retinal arterioles through the activation of GABA type receptors [3], thus, these receptors could be an important
∗ Corresponding author at: 1109 McGaugh Hall, University of California, Irvine, Irvine, CA 92697, USA. Tel.: +1 949 824 6090. E-mail address: [email protected] (J.M. Reyes-Ruiz). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.11.003
target for drugs aimed to improve retinal blood flow in vasculardriven impaired vision. GABA signaling in the retina, and in the central nervous systems of mammals, is mediated by changes of membrane permeability for chloride and bicarbonate ions upon binding of GABA to ionotropic GABAA receptors present in cellular membranes. Some of GABA-elicited responses, as those in postsynaptic neurons, activate and desensitize very fast resulting in what is known as “phasic” inhibition [6]; also, low GABA concentrations in the extracellular space can result in the persistent activation of GABA receptors generating a “tonic” inhibition of excitable cells [6]. GABAA receptors belong to a family of proteins that assemble to form integral ligand gated ion channels (LGICs). Thus far, 19 subunits of this family have been characterized in mammals. The subunits have been
J.M. Reyes-Ruiz et al. / Neuroscience Letters 558 (2014) 78–81
classified in basis of their sequence identity in the subfamilies ␣, , ␥, ␦, , , , and . Most subunits within the GABAA family share at least 20–50% genomic sequence, while in subunits within a subfamily the identity is at least 70% [14]. GABAA receptors can be formed by the combination of different subunits with the major adult isoform comprised of 2 ␣ subunits, 2  subunits and 1 ␥ subunit [20]. There is strong evidence indicating that the 3 subunits of the subfamily are able to form homomeric functional channels, and may form heteromers with subunits from other subfamilies [14]. Homomeric GABA receptors display different pharmacological properties: they are insensitive to bicuculine and to modulation by benzodiazepines [5], are modulated by dopamine and serotonin [12], but most notably, show a slow desensitization after prolonged exposure to the agonist. They were considered a separate type (GABAC ) for many years, but recently they are regarded as part of the GABAA type [13]. Despite the wide use of swine models to study retinal physiology and the important role of GABA receptors in retina function, none of the putative GABA type subunits from pigs has been yet cloned and the pharmacological correspondence between human and pig GABA receptors can only be inferred. Here we used information from the International Swine Genome Sequencing Consortium (SGSC) [2] to clone the pig GABA1 receptor subunit. This clone will aid in the molecular characterization of its pharmacological properties and to gain further insight into this family of receptors in mammals. 2. Materials and methods
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Fig. 1. Amino terminus of the 1 subunit. A stretch of 17 amino acids in the human transcript variant 1 (Hum t1) near the amino terminus is missing in the pig 1 subunit (18 in the rat), but the human transcript variant 2 (Hum t2) share the same amino acidic gap. Overall, the sequence is highly conserved between the three species. The shaded letters highlights the differences with the human sequence. A second transcript variant was cloned from the pig retina (Pig tv) that shared the same splicing site (G40) but the insertion introduced a stop codon after 12 amino acids.
Ensembl automatic analysis pipeline and submitted to UniProtKB under the primary accession number F1S0D1. For this, the remainder of isolated retinal mRNA was reverse-transcripted into cDNA as previously described [16] after confirmation of the presence of transcripts capable of generating functional channels was established by electrophysiology. Primers (Fwd atgttggctgtccagaa; Rev ttatgagaaaatcgacc) were designed using the predicted sequence for the gene reported in the gene bank (NCBI Reference Sequence: XM 001927005.1). 3. Results 3.1. Electrophysiological findings and cloning
Retina from pig was obtained immediately after death from a local slaughterhouse. The collected tissue was snap frozen in dry ice until further use. mRNA was isolated using the Fast Track mRNA isolation kit from Invitrogen. Half of the recovered mRNA was used for microinjections into Xenopus oocytes and after 2–4 days oocytes were tested for expression of neurotransmitter receptors [15], briefly, oocytes were impaled with two microelectrodes filled with 3 M KCl and voltage clamped at −80 mV using a two electrode voltage-clamp amplifier. Oocytes were continuously perfused with gravity-driven frog Ringer’s solution [115 mM NaCl, 2 mM KCl, 1.8 mM CaCl2 , 5 mM Hepes (pH 7.4)] at room temperature (19–21 ◦ C). Data acquisition was performed using WinWCP V 3.9.4 (John Dempster, Glasgow, United Kingdom). The cDNA clone was used as template to generate cRNA as described previously [17] and injected, at a concentration of 0.1 mg/ml, into Xenopus oocytes for further pharmacological analysis.
Oocytes injected with mRNA isolated from pig retina elicited non-desensitizing GABA-induced responses of 8.0 ± 5.3 nA (n = 4 oocytes; mean ± SD, data not shown) and with kinetic characteristics similar as GABA currents from bovine retina [15]. Confirming our previous results in non-injected oocytes where Western blots did not detected endogenous GABA receptors [9], non-injected oocytes also did not respond to GABA even at concentrations as a high as 1 mM [9]. Therefore, the GABA responses in oocytes injected with pig retina mRNA confirmed the presence of full length transcripts for the GABA subunit in the mRNA, and cDNA cloning was performed next. After RT-PCR, a single band of about 1400 bp was obtained and introduced in a vector by T cloning. A sequence of 1389 base pairs with a reading frame of 462 amino acids was confirmed by sequencing. The sequence shared nucleotidic and amino acidic homologies of 91% and 92% with the human and 89% and 91% with the rat (Fig. 1). Three nucleotide substitutions were identified from the predicted sequence (184T > C, 567A > G and 1143C > T, Fig. 2); however, the reported amino acidic sequence was not altered by these nucleotide changes.
2.2. Data analysis
3.2. cDNA clone pharmacological characterization
Concentration/response curve for GABA was built as previously reported [10]. Briefly, a logistic equation of the form I(x) = Imin + (Imax − Imin )/[1 + (x/EC50)k], where x is the concentration of GABA (in M), I is the amplitude of the GABA-induced current (in nA), and k is the slope of the curve, was fitted to the experimental data (SigmaPlot 10). Experimental data are shown as mean ± SD unless otherwise stated. The rise to maximum was measured between the 10–90% of the ion current activation using clampfit 10.2 (Axon instruments).
Oocytes injected with synthetic mRNA generated from the cDNA clone of the pig retina, expressed membrane receptors, highly sensitive for GABA, with an EC50 of 698 ± 489 nM and a Hill coefficient of 2.6 ± 0.2 (mean ± sd; n = 5; Fig. 3). GABA currents rate of activation accelerated with the concentration, from a rise to maximum of 67.8 ± 14.2 s at 0.3 M GABA to 4.5 ± 1.5 s at 3 M
2.1. Functional expression
2.3. cDNA cloning. After the recent completion of the draft sequencing of the pig genome by the SGSC [2], many ESTs have been reported and are now available for molecular biology analyses [21]. We proceeded to clone the putative pig GABA1 receptor subunit identified by an
Fig. 2. Schematic gene structure of predicted (A) and cloned sequences (B and C). (A) Gene bank sequence XM 001927005.1, predicted GABA 1 subunit from the pig. (B) Cloned sequence, functionally active. Vertical lines represent the 3 nucleotidic substitutions found. (C) Transcript variant. Black rectangle illustrates a 140 bp insertion that introduces an early stop codon (arrow).
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intracellular loop between TM3 and TM4 [19] and therefore conserved too. 4. Discussion
Fig. 3. Concentration dependence of pig GABA1. Left, ion currents elicited by different concentrations of GABA in an oocyte voltage clamped at −80 mV. Horizontal lines above the responses, here and in the next figure, indicate time of substance applications. Right, plot of normalized maximum amplitude of responses induced by incremental concentrations of GABA. Standard deviation bars are smaller than the symbols at low concentrations of GABA. Solid curve is the fit of the Hill equation to experimental data.
GABA (mean ± sd; n = 5). The maximum amplitude (5.2 ± 1.9 A; mean ± sd; n = 5) was reached around 3 M. At concentrations of GABA larger than 1 M the responses showed a slow desensitization that was not further investigated. A basic analysis of the pharmacological properties of the receptor indicated that GABA currents were not affected by high concentrations of bicuculline or pentobarbital (Fig. 4); however, they were negatively modulated by dopamine and (1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid; (TPMPA), a specific antagonist of GABA receptors in humans and rats. This data indicated that the general properties of the pig GABA receptor are very similar to those reported for the rat and human GABA1 subunit. The most notable difference found in the pig receptor was the absence of a stretch of 17 amino acids near the amino terminus (R41–V58) conserved in the rat and the human (Fig. 1). The ligand binding site for this receptor has been placed between Y104 and Y247 [18] and therefore the lack of these amino acids does not affect GABA binding, as it is confirmed by the functional expression of the receptor. The sequence here reported has a higher match with the human GABA1 subunit, transcript variant 2 (NCBI Reference Sequence: NM 001256703.1) which lacks an in-frame exon [11]. Out of 27 clones tested, 24 contained this sequence, and 3 were 140 bp longer (Fig. 2). The extra nucleotides were inserted in the splicing site, but a stop codon was found 12 amino acids downstream (Figs. 1 and 2). Furthermore, the 140 nucleotides were subjected to BLAST analysis [1] and no significant similarities to any known gene were found. The last 199 amino acids, which account for the 4 TMs, are conserved with an identity of 96%. Phosphorylation sites are in the
The pig is the most consumed animal in the world and genetic stocks improvements are underway [4], moreover, the physiological and anatomical similitudes between human and pig have made of the swine model one of the preferred animal models in vision research [7]. For these reasons, the knowledge of its genetics is important and the SGSC has made a great effort which has been rewarded with the release of hundreds of thousands ESTs [21]. Among those, studying ESTs for proteins from the CNS helps to widen the understanding of neurotransmitter receptors and shed light in how the evolution of these genes among mammals has occurred. Although we anticipated finding the 1 subunit of the GABA receptor abundantly in the retina of the pig, and that the pharmacological properties would be similar to 1 subunits from the human and other mammals that have been characterized, it was unexpected to find that the most abundant transcript was the human equivalent to transcription variant 2. This variant was cloned and pharmacologically characterized in humans by Martinez-Torres et al. [11], but has not been characterized in other species so far; although it is found in the gene bank among predicted sequences of many other organisms. However, it seems to be the most abundant in the pig. Actually, the “full length” variant was not found at all, which could mean that this is the main variant in this species. It is important to note that we only tested the retina from 1 specimen, and screening more cDNA libraries from other pigs will be necessary to confirm that this variant is the most abundant indeed. It is worth noting that the predicted sequence in the gene bank for the pig subunit is this same transcript and no “full length” sequence is yet available. We were able to clone an apparent second transcript variant in the pig, but since the inserted sequence did not translate into a functional protein and that the sequence did not BLAST to any other known functional protein, it is very likely that it is only a stretch of intron that survived the splicing. There are 4 transcript variants reported in human for this protein. More extensive cloning efforts will be needed to confirm their existence in the pig. Role of the funding source Funding source was not involved in the design or decisions taken in this project.
Fig. 4. Properties of pig GABA1 receptors. Left, current responses elicited by GABA and its modulation by bicuculline (Bic), dopamine (DA), (1,2,5,6-tetrahydropyridin4-yl)methylphosphinic acid; (TPMPA) and pentobarbital (Pento). Currents are shown normalized for better comparison. Right, plot of the mean ± SD of the effect of the compounds shown at the left, n = 5.
J.M. Reyes-Ruiz et al. / Neuroscience Letters 558 (2014) 78–81
Author contributions All authors had full access to the data and take responsibility for the integrity and accuracy of the data analysis. Study concept and design: JMRR, AL, RM. Acquisition and analysis of data, manuscript draft and statistical analysis: JMRR, AL, RM. Obtained funding: RM. Administrative, technical, and material support: RM. Study supervision: JMRR, AL and RM. Conflict of interest Authors state no conflict of interest. Acknowledgements This work was supported in part by King Abdul Aziz City for Science and Technology (Saudi Arabia) Grant KACST-46749 (to R.M.). Authors also wish to acknowledge Manuel Candelario Sr. and Jr. for their help in this project. References [1] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment search tool, J. Mol. Biol. 215 (1990) 403–410. [2] A.L. Archibald, L. Bolund, C. Churcher, M. Fredholm, M.A. Groenen, B. Harlizius, K.T. Lee, D. Milan, J. Rogers, M.F. Rothschild, H. Uenishi, J. Wang, L.B. Schook, Pig genome sequence-analysis and publication strategy, BMC Genom. 11 (2010) 438. [3] T. Bek, K. Holmgaard, GABA-induced relaxation of porcine retinal arterioles in vitro depends on inhibition from the perivascular retina and is mediated by GABAC receptors, Invest. Ophthalmol. Vis. Sci. 53 (2012) 3309–3315. [4] J.C.M. Dekkers, P.K. Mathur, E.F. Knol, Genetic improvement of the pig, in: M.F. Rothschild, A. Ruvinsky, Wallingford (Eds.), The Genetics of the Pig, 2nd ed., CAB International, 2011, pp. 390–425. [5] M.G. Darlison, I. Pahal, C. Thode, Consequences of the evolution of the GABA(A) receptor gene family, Cell. Mol. Neurobiol. 25 (2005) 607–624.
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[6] M. Farrant, Z. Nusser, Variations on an inhibitory theme: phasic and tonic activation of GABAA receptors, Nat. Rev. Neurosci. 6 (2005) 215–229. [7] C.G. Gerke, Y. Hao, F. Wong, Topography of rods and cones in the retina of the domestic pig, HKMJ 1 (1995) 302–308. [8] A. Hendrickson, D. Hicks, Distribution and density of medium- and shortwavelength selective cones in the domestic pig retina, Exp. Eye Res. 74 (2002) 435–444. [9] A. Limon, J.L. Gallegos-Perez, J.M. Reyes-Ruiz, M.A. Aljohi, A.S. Alshanqeeti, R. Miledi, The endogenous GABA bioactivity of camel, bovine, goat and human milks, Food Chem. 145 (2014) 481–487. [10] A. Limon, J.M. Reyes-Ruiz, R.G. Vaswani, A.R. Chamberlin, R. Miledi, Kaitocephalin antagonism of glutamate receptors expressed in Xenopus oocytes, ACS Chem. Neurosci. 1 (2010) 175–181. [11] A. Martínez-Torres, A.E. Vazquez, M.M. Panicker, R. Miledi, Cloning and functional expression of alternative spliced variants of the rho1 gammaaminobutyrate receptor, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 4019–4022. [12] L.D. Ochoa-de la Paz, A. Estrada-Mondragón, A. Limón, R. Miledi, A. MartínezTorres, Dopamine and serotonin modulate human GABA1 receptors expressed in Xenopus laevis oocytes, ACS Chem. Neurosci. 3 (2012) 96–104. [13] R.W. Olsen, W. Sieghart, International union of pharmacology. LXX. Subtypes of ␥-aminobutyric acid A receptors: classification on the basis of subunit composition, pharmacology, and function. Update, Pharmacol. Rev. 60 (2008) 243–260. [14] R.W. Olsen, A.J. Tobin, Molecular biology of GABAA receptors, FASEB J. 4 (1990) 1469–1480. [15] L. Polenzani, R.M. Woodward, R. Miledi, Expression of mammalian gammaaminobutyric acid receptors with distinct pharmacology in Xenopus oocytes, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 4318–4322. [16] J.M. Reyes-Ruiz, A. Limon, M.J. Korn, P.A. Nakamura, N.J. Shirkey, J.K. Wong, R. Miledi, Profiling neurotransmitter receptor expression in the Ambystoma mexicanum brain, Neurosci. Lett. 538 (2013) 32–37. [17] J.M. Reyes-Ruiz, L.D. Ochoa-de la Paz, A. Martínez-Torres, R. Miledi, Functional impact of serial deletions at the C-terminus of the human GABArho1 receptor, Biochim. Biophys. Acta 1798 (2010) 1002–1007. [18] A. Sedelnikova, C.D. Smith, S.O. Zakharkin, D. Davis, D.S. Weiss, Y. Chang, Mapping the rho1 GABA(C) receptor agonist binding pocket. Constructing a complete model, J. Biol. Chem. 280 (2005) 1535–1542. [19] A. Sedelnikova, D.S. Weiss, Phosphorylation of the recombinant rho1 GABA receptor, Int. J. Dev. Neurosci. 20 (2002) 237–246. [20] E. Sigel, M.E. Steinmann, Structure, function, and modulation of GABA(A) receptors, J. Biol. Chem. 287 (2012) 40224–40231. [21] H. Uenishi, T. Morozumi, D. Toki, T. Eguchi-Ogawa, L.A. Rund, L.B. Schook, Large-scale sequencing based on full-length-enriched cDNA libraries in pigs: contribution to annotation of the pig genome draft sequence, BMC Genom. 13 (2012) 581.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Cloning and characterization of the ionotropic GABA receptor subunit 1 from pig (Sus scrofa) Jorge Mauricio Reyes-Ruiz ∗ , Agenor Limon, Ricardo Miledi Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA 92697, USA
h i g h l i g h t s • • • • •
Oocytes injected with retina mRNA generated slow desensitizing GABA currents. A cDNA clone was obtained by RT-PCR and cRNA generated from it. Xoocytes injected with cRNA were used for pharmacological characterization. The cloned nucleotidic sequence is similar to the human transcription variant 2. A 2nd splicing variant was cloned with a stop codon early in the reading frame.
a r t i c l e
i n f o
Article history: Received 5 September 2013 Received in revised form 28 October 2013 Accepted 3 November 2013 Keywords: Xenopus oocyte Electrophysiology mRNA Sequencing ESTs
a b s t r a c t Since human and pig eyes have remarkably anatomical and physiological similitudes swine models have been broadly used for functional studies and therapeutic research. Recently, a GABA-mediated relaxation of retinal vascularity suggested that GABA signaling may be used to improve retinal blood flow in vascular-driven impaired vision, and a further molecular characterization of GABA receptors would be beneficial. However, none of the GABA type subunits from pigs has been yet cloned; Among the 19 subunits that compose the family of GABAA receptors, 1–3 subunits are capable of forming homomeric channels. These homomeric receptors are particularly interesting because their pharmacological and kinetic properties are notably different from receptors composed by other GABAA subunits. Here we report the cloning of the GABA1subunit from the pig and the functional expression of homomeric channels in Xenopus oocytes. The most notable difference found in the pig GABA1 receptor was the absence of a stretch of 17 amino acids near the amino terminus (R41–V58) conserved in the rat and the human. This sequence has a higher nucleotidic match with the transcript variant 2 of the human GABA1 subunit. Xenopus oocytes injected with cRNA from the receptor generated currents when exposed to GABA that shared all the characteristics of other GABA1 subunits in mammals, including its modulation by dopamine. This study will help to increase the knowledge of the genetics of the pig, further the understanding of this important neurotransmitter receptor family and will shed some light in the evolution of these genes among mammals. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Human and pig eyes have remarkably anatomical and physiological similitudes [7,8]; therefore, swine models are preferable to murine models for ontogeny studies and therapeutic research. Recently it was shown that ␥-aminobutyrate (GABA) participates in the relaxation of retinal arterioles through the activation of GABA type receptors [3], thus, these receptors could be an important
∗ Corresponding author at: 1109 McGaugh Hall, University of California, Irvine, Irvine, CA 92697, USA. Tel.: +1 949 824 6090. E-mail address: [email protected] (J.M. Reyes-Ruiz). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.11.003
target for drugs aimed to improve retinal blood flow in vasculardriven impaired vision. GABA signaling in the retina, and in the central nervous systems of mammals, is mediated by changes of membrane permeability for chloride and bicarbonate ions upon binding of GABA to ionotropic GABAA receptors present in cellular membranes. Some of GABA-elicited responses, as those in postsynaptic neurons, activate and desensitize very fast resulting in what is known as “phasic” inhibition [6]; also, low GABA concentrations in the extracellular space can result in the persistent activation of GABA receptors generating a “tonic” inhibition of excitable cells [6]. GABAA receptors belong to a family of proteins that assemble to form integral ligand gated ion channels (LGICs). Thus far, 19 subunits of this family have been characterized in mammals. The subunits have been
J.M. Reyes-Ruiz et al. / Neuroscience Letters 558 (2014) 78–81
classified in basis of their sequence identity in the subfamilies ␣, , ␥, ␦, , , , and . Most subunits within the GABAA family share at least 20–50% genomic sequence, while in subunits within a subfamily the identity is at least 70% [14]. GABAA receptors can be formed by the combination of different subunits with the major adult isoform comprised of 2 ␣ subunits, 2  subunits and 1 ␥ subunit [20]. There is strong evidence indicating that the 3 subunits of the subfamily are able to form homomeric functional channels, and may form heteromers with subunits from other subfamilies [14]. Homomeric GABA receptors display different pharmacological properties: they are insensitive to bicuculine and to modulation by benzodiazepines [5], are modulated by dopamine and serotonin [12], but most notably, show a slow desensitization after prolonged exposure to the agonist. They were considered a separate type (GABAC ) for many years, but recently they are regarded as part of the GABAA type [13]. Despite the wide use of swine models to study retinal physiology and the important role of GABA receptors in retina function, none of the putative GABA type subunits from pigs has been yet cloned and the pharmacological correspondence between human and pig GABA receptors can only be inferred. Here we used information from the International Swine Genome Sequencing Consortium (SGSC) [2] to clone the pig GABA1 receptor subunit. This clone will aid in the molecular characterization of its pharmacological properties and to gain further insight into this family of receptors in mammals. 2. Materials and methods
79
Fig. 1. Amino terminus of the 1 subunit. A stretch of 17 amino acids in the human transcript variant 1 (Hum t1) near the amino terminus is missing in the pig 1 subunit (18 in the rat), but the human transcript variant 2 (Hum t2) share the same amino acidic gap. Overall, the sequence is highly conserved between the three species. The shaded letters highlights the differences with the human sequence. A second transcript variant was cloned from the pig retina (Pig tv) that shared the same splicing site (G40) but the insertion introduced a stop codon after 12 amino acids.
Ensembl automatic analysis pipeline and submitted to UniProtKB under the primary accession number F1S0D1. For this, the remainder of isolated retinal mRNA was reverse-transcripted into cDNA as previously described [16] after confirmation of the presence of transcripts capable of generating functional channels was established by electrophysiology. Primers (Fwd atgttggctgtccagaa; Rev ttatgagaaaatcgacc) were designed using the predicted sequence for the gene reported in the gene bank (NCBI Reference Sequence: XM 001927005.1). 3. Results 3.1. Electrophysiological findings and cloning
Retina from pig was obtained immediately after death from a local slaughterhouse. The collected tissue was snap frozen in dry ice until further use. mRNA was isolated using the Fast Track mRNA isolation kit from Invitrogen. Half of the recovered mRNA was used for microinjections into Xenopus oocytes and after 2–4 days oocytes were tested for expression of neurotransmitter receptors [15], briefly, oocytes were impaled with two microelectrodes filled with 3 M KCl and voltage clamped at −80 mV using a two electrode voltage-clamp amplifier. Oocytes were continuously perfused with gravity-driven frog Ringer’s solution [115 mM NaCl, 2 mM KCl, 1.8 mM CaCl2 , 5 mM Hepes (pH 7.4)] at room temperature (19–21 ◦ C). Data acquisition was performed using WinWCP V 3.9.4 (John Dempster, Glasgow, United Kingdom). The cDNA clone was used as template to generate cRNA as described previously [17] and injected, at a concentration of 0.1 mg/ml, into Xenopus oocytes for further pharmacological analysis.
Oocytes injected with mRNA isolated from pig retina elicited non-desensitizing GABA-induced responses of 8.0 ± 5.3 nA (n = 4 oocytes; mean ± SD, data not shown) and with kinetic characteristics similar as GABA currents from bovine retina [15]. Confirming our previous results in non-injected oocytes where Western blots did not detected endogenous GABA receptors [9], non-injected oocytes also did not respond to GABA even at concentrations as a high as 1 mM [9]. Therefore, the GABA responses in oocytes injected with pig retina mRNA confirmed the presence of full length transcripts for the GABA subunit in the mRNA, and cDNA cloning was performed next. After RT-PCR, a single band of about 1400 bp was obtained and introduced in a vector by T cloning. A sequence of 1389 base pairs with a reading frame of 462 amino acids was confirmed by sequencing. The sequence shared nucleotidic and amino acidic homologies of 91% and 92% with the human and 89% and 91% with the rat (Fig. 1). Three nucleotide substitutions were identified from the predicted sequence (184T > C, 567A > G and 1143C > T, Fig. 2); however, the reported amino acidic sequence was not altered by these nucleotide changes.
2.2. Data analysis
3.2. cDNA clone pharmacological characterization
Concentration/response curve for GABA was built as previously reported [10]. Briefly, a logistic equation of the form I(x) = Imin + (Imax − Imin )/[1 + (x/EC50)k], where x is the concentration of GABA (in M), I is the amplitude of the GABA-induced current (in nA), and k is the slope of the curve, was fitted to the experimental data (SigmaPlot 10). Experimental data are shown as mean ± SD unless otherwise stated. The rise to maximum was measured between the 10–90% of the ion current activation using clampfit 10.2 (Axon instruments).
Oocytes injected with synthetic mRNA generated from the cDNA clone of the pig retina, expressed membrane receptors, highly sensitive for GABA, with an EC50 of 698 ± 489 nM and a Hill coefficient of 2.6 ± 0.2 (mean ± sd; n = 5; Fig. 3). GABA currents rate of activation accelerated with the concentration, from a rise to maximum of 67.8 ± 14.2 s at 0.3 M GABA to 4.5 ± 1.5 s at 3 M
2.1. Functional expression
2.3. cDNA cloning. After the recent completion of the draft sequencing of the pig genome by the SGSC [2], many ESTs have been reported and are now available for molecular biology analyses [21]. We proceeded to clone the putative pig GABA1 receptor subunit identified by an
Fig. 2. Schematic gene structure of predicted (A) and cloned sequences (B and C). (A) Gene bank sequence XM 001927005.1, predicted GABA 1 subunit from the pig. (B) Cloned sequence, functionally active. Vertical lines represent the 3 nucleotidic substitutions found. (C) Transcript variant. Black rectangle illustrates a 140 bp insertion that introduces an early stop codon (arrow).
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J.M. Reyes-Ruiz et al. / Neuroscience Letters 558 (2014) 78–81
intracellular loop between TM3 and TM4 [19] and therefore conserved too. 4. Discussion
Fig. 3. Concentration dependence of pig GABA1. Left, ion currents elicited by different concentrations of GABA in an oocyte voltage clamped at −80 mV. Horizontal lines above the responses, here and in the next figure, indicate time of substance applications. Right, plot of normalized maximum amplitude of responses induced by incremental concentrations of GABA. Standard deviation bars are smaller than the symbols at low concentrations of GABA. Solid curve is the fit of the Hill equation to experimental data.
GABA (mean ± sd; n = 5). The maximum amplitude (5.2 ± 1.9 A; mean ± sd; n = 5) was reached around 3 M. At concentrations of GABA larger than 1 M the responses showed a slow desensitization that was not further investigated. A basic analysis of the pharmacological properties of the receptor indicated that GABA currents were not affected by high concentrations of bicuculline or pentobarbital (Fig. 4); however, they were negatively modulated by dopamine and (1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid; (TPMPA), a specific antagonist of GABA receptors in humans and rats. This data indicated that the general properties of the pig GABA receptor are very similar to those reported for the rat and human GABA1 subunit. The most notable difference found in the pig receptor was the absence of a stretch of 17 amino acids near the amino terminus (R41–V58) conserved in the rat and the human (Fig. 1). The ligand binding site for this receptor has been placed between Y104 and Y247 [18] and therefore the lack of these amino acids does not affect GABA binding, as it is confirmed by the functional expression of the receptor. The sequence here reported has a higher match with the human GABA1 subunit, transcript variant 2 (NCBI Reference Sequence: NM 001256703.1) which lacks an in-frame exon [11]. Out of 27 clones tested, 24 contained this sequence, and 3 were 140 bp longer (Fig. 2). The extra nucleotides were inserted in the splicing site, but a stop codon was found 12 amino acids downstream (Figs. 1 and 2). Furthermore, the 140 nucleotides were subjected to BLAST analysis [1] and no significant similarities to any known gene were found. The last 199 amino acids, which account for the 4 TMs, are conserved with an identity of 96%. Phosphorylation sites are in the
The pig is the most consumed animal in the world and genetic stocks improvements are underway [4], moreover, the physiological and anatomical similitudes between human and pig have made of the swine model one of the preferred animal models in vision research [7]. For these reasons, the knowledge of its genetics is important and the SGSC has made a great effort which has been rewarded with the release of hundreds of thousands ESTs [21]. Among those, studying ESTs for proteins from the CNS helps to widen the understanding of neurotransmitter receptors and shed light in how the evolution of these genes among mammals has occurred. Although we anticipated finding the 1 subunit of the GABA receptor abundantly in the retina of the pig, and that the pharmacological properties would be similar to 1 subunits from the human and other mammals that have been characterized, it was unexpected to find that the most abundant transcript was the human equivalent to transcription variant 2. This variant was cloned and pharmacologically characterized in humans by Martinez-Torres et al. [11], but has not been characterized in other species so far; although it is found in the gene bank among predicted sequences of many other organisms. However, it seems to be the most abundant in the pig. Actually, the “full length” variant was not found at all, which could mean that this is the main variant in this species. It is important to note that we only tested the retina from 1 specimen, and screening more cDNA libraries from other pigs will be necessary to confirm that this variant is the most abundant indeed. It is worth noting that the predicted sequence in the gene bank for the pig subunit is this same transcript and no “full length” sequence is yet available. We were able to clone an apparent second transcript variant in the pig, but since the inserted sequence did not translate into a functional protein and that the sequence did not BLAST to any other known functional protein, it is very likely that it is only a stretch of intron that survived the splicing. There are 4 transcript variants reported in human for this protein. More extensive cloning efforts will be needed to confirm their existence in the pig. Role of the funding source Funding source was not involved in the design or decisions taken in this project.
Fig. 4. Properties of pig GABA1 receptors. Left, current responses elicited by GABA and its modulation by bicuculline (Bic), dopamine (DA), (1,2,5,6-tetrahydropyridin4-yl)methylphosphinic acid; (TPMPA) and pentobarbital (Pento). Currents are shown normalized for better comparison. Right, plot of the mean ± SD of the effect of the compounds shown at the left, n = 5.
J.M. Reyes-Ruiz et al. / Neuroscience Letters 558 (2014) 78–81
Author contributions All authors had full access to the data and take responsibility for the integrity and accuracy of the data analysis. Study concept and design: JMRR, AL, RM. Acquisition and analysis of data, manuscript draft and statistical analysis: JMRR, AL, RM. Obtained funding: RM. Administrative, technical, and material support: RM. Study supervision: JMRR, AL and RM. Conflict of interest Authors state no conflict of interest. Acknowledgements This work was supported in part by King Abdul Aziz City for Science and Technology (Saudi Arabia) Grant KACST-46749 (to R.M.). Authors also wish to acknowledge Manuel Candelario Sr. and Jr. for their help in this project. References [1] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment search tool, J. Mol. Biol. 215 (1990) 403–410. [2] A.L. Archibald, L. Bolund, C. Churcher, M. Fredholm, M.A. Groenen, B. Harlizius, K.T. Lee, D. Milan, J. Rogers, M.F. Rothschild, H. Uenishi, J. Wang, L.B. Schook, Pig genome sequence-analysis and publication strategy, BMC Genom. 11 (2010) 438. [3] T. Bek, K. Holmgaard, GABA-induced relaxation of porcine retinal arterioles in vitro depends on inhibition from the perivascular retina and is mediated by GABAC receptors, Invest. Ophthalmol. Vis. Sci. 53 (2012) 3309–3315. [4] J.C.M. Dekkers, P.K. Mathur, E.F. Knol, Genetic improvement of the pig, in: M.F. Rothschild, A. Ruvinsky, Wallingford (Eds.), The Genetics of the Pig, 2nd ed., CAB International, 2011, pp. 390–425. [5] M.G. Darlison, I. Pahal, C. Thode, Consequences of the evolution of the GABA(A) receptor gene family, Cell. Mol. Neurobiol. 25 (2005) 607–624.
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