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Isovaline Does Not Activate GABAB Receptor-Coupled Potassium

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Isovaline Does Not Activate GABAB Receptor-Coupled Potassium RESEARCH ARTICLE Isovaline Does Not Activate GABAB Receptor- Coupled Potassium Currents in GABAB Expressing AtT-20 Cells and Cultured Rat Hippocampal Neurons Kimberley A. Pitman1,2, Stephanie L. Borgland2*, Bernard MacLeod1, Ernest Puil1 1 Department of Anesthesiology, Pharmacology & Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada, 2 Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada * [email protected] Abstract Isovaline is a non-proteinogenic amino acid that has analgesic properties. R-isovaline is a proposed agonist of the γ-aminobutyric acid type B (GABAB) receptor in the thalamus and OPEN ACCESS peripheral tissue. Interestingly, the responses to R-isovaline differ from those of the canoni- Citation: Pitman KA, Borgland SL, MacLeod B, Puil cal GABAB receptor agonist R-baclofen, warranting further investigation. Using whole cell E (2015) Isovaline Does Not Activate GABA B recording techniques we explored isovaline actions on GABAB receptors coupled to Receptor-Coupled Potassium Currents in GABA B rectifying K+ channels in cells of recombinant and native receptor preparations. In AtT-20 Expressing AtT-20 Cells and Cultured Rat Hippocampal Neurons. PLoS ONE 10(2): e0118497. cells transfected with GABAB receptor subunits, bath application of the GABAB receptor doi:10.1371/journal.pone.0118497 agonists, GABA (1 μM) and R-baclofen (5 μM) produced inwardly rectifying currents that + μ Academic Editor: Steven Barnes, Dalhousie reversed approximately at the calculated reversal potential for K R- isovaline (50 Mto1mM) University, CANADA and S-isovaline (500 μM) did not evoke a current. R-isovaline applied either extracellularly μ μ μ Received: November 26, 2014 (250 M) or intracellularly (10 M) did not alter responses to GABA at 1 M. Co-administra- tion of R-isovaline (250 μM) with a low concentration (10 nM) of GABA did not result in a re- Accepted: January 19, 2015 sponse. In cultured rat hippocampal neurons that natively express GABAB receptors, Published: February 23, 2015 R-baclofen (5 μM) induced GABAB receptor-dependent inward currents. Under the same Copyright: © 2015 Pitman et al. This is an open conditions R-isovaline (1 or 50 μM) did not evoke a current or significantly alter R-baclofen- access article distributed under the terms of the induced effects. Therefore, R-isovaline does not interact with recombinant or native GABA Creative Commons Attribution License, which permits B + unrestricted use, distribution, and reproduction in any receptors to open K channels in these preparations. medium, provided the original author and source are credited. Data Availability Statement: Data are available from Figshare at the following URL: http://figshare.com/s/ 37f4cd8a97b711e4ab1006ec4bbcf141. Funding: These studies were supported by the Jean Introduction Hugill Anesthesia Research Endowment to B.M and γ Receptors for -aminobutyric acid (GABA), especially of the GABAB type, are a promising tar- E.P. S.L.B. is supported by a Canadian Institutes of get in analgesia. However, the use of the prototypical GABA agonist, baclofen, is limited due Health Research New Investigator Award (MOP- B 104357). The funders had no role in study design, to the development of severe side effects [1]. Isovaline is an unusual non-proteinogenic amino data collection and analysis, decision to publish, or acid that is anti-nociceptive without side effects typical of GABAB agonists [2,3]. A component preparation of the manuscript. of the analgesia produced by isovaline is attributable to activation of GABAB receptors [2]. PLOS ONE | DOI:10.1371/journal.pone.0118497 February 23, 2015 1/13 Isovaline Does Not Modulate GABAB Receptors Competing Interests: B.M and E.P have a patent GABAB receptors are obligate heterodimers, made up of a GABAB1 and a GABAB2 subunit application titled “Isovaline for the treatment of pain” – [4 6]. Surprisingly, there are no pharmacologically distinct GABAB receptor subtypes [7,8]. (Patent number: US8278355 B2). E.P. is the chief Critical residues for orthosteric agonist and antagonist binding are located on the GABAB1 sub- executive officer and B.M is on the board of directors – of TherExcell Pharma Inc. This does not alter the unit within a region known as the Venus flytrap domain [9 11], while allosteric modulators of authors' adherence to PLOS ONE policies on sharing the GABAB receptor act at sites on the GABAB2 subunit [12]. GABAB receptors are G-protein data and materials. coupled receptors that associate with the pertussis toxin sensitive Gαi/o family of G-proteins [13]. Conventional cellular effects of GABAB receptor agonism include inhibition of adenylate + cyclase, Gβγ-mediated activation of G-protein coupled inwardly rectifying K (GIRK) channels 2+ and Gβγ-mediated inhibition of voltage gated Ca channels [8,14,15]. ’ Isovaline s action at GABAB receptors is atypical as demonstrated in thalamic neurons of brain slices. Compared to R-baclofen, R-isovaline-evoked K+ currents are slow in onset and long lasting [16,17]. Also in contrast to R-baclofen, the response to R-isovaline is blocked by pre-application of a GABAB receptor antagonist, but not by application of the GABAB antago- nist subsequent to the initiation of the K+ current [16]. Furthermore, some neurons that re- spond to R-baclofen do not respond to R-isovaline [16]. The ability of R-isovaline to activate currents in sensitive neurons does not appear to result from GABA release and subsequent + postsynaptic activation of GABAB-mediated K currents, since inhibition of other GABA-me- diated currents does not alter R-isovaline responses [16]. Here we test the effects of R-isovaline on GABAB receptors in a neuronal expression system as well as in isolated hippocampal neu- rons natively expressing GABAB receptors. Materials and Methods AtT-20 cell culturing, transfection and electrophysiology Mouse pituitary AtT-20 cells [18,19] obtained from Dr. C. Chavkin’s laboratory at the Univer- sity of Washington (March, 2010) were grown in Gibco high glucose Dulbecco’s modified Eagle medium (Invitrogen) with 1% fetal bovine Serum, 10% horse serum, penicillin-strepto- mycin and 0.2 mM L-glutamine and kept in an incubator at 37°C with 5% CO2. Cells were pas- saged every 2–3 days. For transient transfection cells were plated in a 35mm culture dish at 90% confluency and left for 24 hours. Cells were then co-transfected overnight in serum con- taining media using Lipofectamine 2000 (Invitrogen, Life technologies, Burlington, ON) fol- ’ lowing the manufacturer s protocols. Cells were co-transfected with cDNA for the GABAB1a and GABAB2 subunits as well as green fluorescent protein (GFP) to aid identification of trans- fected cells (1:2:5 ratio of GFP:GABAB1a:GABAB2). The following day the transfection media were removed and cells were re-plated onto poly-D-lysine coated glass cover slips which were then incubated for a further 24 hours before use. To confirm membrane expression of the GABAB receptor in cells that were suitable for patching (isolated from one another), we performed immunohistochemistry for the GABAB1 subunit which is unable to traffic to the membrane without associating with a GABAB2 subunit [4–6]. Cells were fixed with 4% formaldehyde for 30 minutes then blocked with 0.5% bovine serum albumin for 1 hour. Cells were incubated with or without mouse anti-GABAB1 (1:1000 dilution, ab55051, Abcam, Toronto) overnight at 4°C. Goat anti-mouse conjugated to Alexa Fluor 546 (Life technologies) was applied at a 1:200 dilution for 1 hour at room temperature. DAPI (4',6-diamidino-2-phenylindole) was applied at 1:10,000 dilution for 5 minutes to stain the nuclei. Coverslips were washed with phosphate buffered saline 3 times between each step. The glass coverslips were mounted onto glass slides and left to dry for at least 72 hours. Confo- cal microscopy was performed using an Olympus FluoView FV1000 confocal microscope with a 60x objective (Tokyo, Japan). PLOS ONE | DOI:10.1371/journal.pone.0118497 February 23, 2015 2/13 Isovaline Does Not Modulate GABAB Receptors For electrophysiological recordings, cells were removed from the incubator and the medium was replaced at room temperature with high K+ extracellular solution containing (in mM): – μ NaCl (130), KCL (20), CaCl2 (2), MgCl2 (1), HEPES (10), glucose (35), and 0.1 1 M tetrodo- toxin. The solution had a pH = 7.4 (adjusted with NaOH) and an osmolality of 330 ± 5 mOsm. The glass coverslip was cut into sections and placed into a 50 μL fast exchange diamond bath (Warner Instruments, Hamden, CT) constantly perfused (*2 ml/min) with extracellular solution. Recording electrodes were made out of thin-walled borosilicate glass (World Preci- sion Instruments, Sarasota, FL) and had a resistance between 3–5MO when filled with an in- tracellular pipette solution containing (in mM): K-gluconate (130), NaCl (20), MgCl2 (1), EGTA (10), glucose (10), HEPES (10), MgATP (5) and Na3GTP (0.1) with pH = 7.2 (NaOH/ HCl) and an osmolality of 310 ± 3 mOsm. The calculated Nernst equilibrium potential for K+ was approximately -47 mV. Cells were visualized with an Axiovert 25 inverted fluorescent mi- croscope (Zeiss, Germany). Whole cell recordings from fluorescing cells were performed using a List EPC 7 amplifier (HEKA, Germany). Recordings were filtered at 3 kHz, digitized at 10 kHz and analysed using pClamp 8.2 software (Molecular Devices, Sunnyvale, CA). Cells were voltage-clamped at -50 mV. The current-voltage (IV) relationship was measured each minute by applying a family of 400 ms voltage steps, from -100 mV to +20 mV, in 10 mV increments. A junction potential of -12.5 mV was accounted for offline. The current was measured as the average current in the last 100 ms of each voltage step. In all experiments except one, drugs were applied via the bath for 2–5 min, and then washed off. In one experiment R-isovaline was added into the intracellular pipette solution.
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