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Evidence for phosphorylation‐dependent internalization of recombinant human ρ1 GABA C receptors

1999; Wiley; Volume: 518; Issue: 2 Linguagem: Inglês

10.1111/j.1469-7793.1999.0385p.x

1469-7793

Natalia Filippova, R.K. Dudley, David S. Weiss,

Nicotinic Acetylcholine Receptors Study

The Journal of PhysiologyVolume 518, Issue 2 p. 385-399 Free Access Evidence for phosphorylation-dependent internalization of recombinant human ρ1 GABAC receptors Natalia Filippova, Natalia Filippova Department of Neurobiology, University of Alabama at Birmingham School of Medicine, 1719 Sixth Avenue South, CIRC 410, Birmingham, AL 35294-0021, USASearch for more papers by this authorRichard Dudley, Richard Dudley Department of Neurobiology, University of Alabama at Birmingham School of Medicine, 1719 Sixth Avenue South, CIRC 410, Birmingham, AL 35294-0021, USASearch for more papers by this authorDavid S. Weiss, Corresponding Author David S. Weiss Department of Neurobiology, University of Alabama at Birmingham School of Medicine, 1719 Sixth Avenue South, CIRC 410, Birmingham, AL 35294-0021, USACorresponding author D. S. Weiss: Department of Neurobiology, University of Alabama at Birmingham School of Medicine, 1719 Sixth Avenue So., CIRC 410, Birmingham, AL 35294-0021, USA. Email: dweiss@nrc.uab.eduSearch for more papers by this author Natalia Filippova, Natalia Filippova Department of Neurobiology, University of Alabama at Birmingham School of Medicine, 1719 Sixth Avenue South, CIRC 410, Birmingham, AL 35294-0021, USASearch for more papers by this authorRichard Dudley, Richard Dudley Department of Neurobiology, University of Alabama at Birmingham School of Medicine, 1719 Sixth Avenue South, CIRC 410, Birmingham, AL 35294-0021, USASearch for more papers by this authorDavid S. Weiss, Corresponding Author David S. Weiss Department of Neurobiology, University of Alabama at Birmingham School of Medicine, 1719 Sixth Avenue South, CIRC 410, Birmingham, AL 35294-0021, USACorresponding author D. S. Weiss: Department of Neurobiology, University of Alabama at Birmingham School of Medicine, 1719 Sixth Avenue So., CIRC 410, Birmingham, AL 35294-0021, USA. Email: dweiss@nrc.uab.eduSearch for more papers by this author First published: 07 September 2004 https://doi.org/10.1111/j.1469-7793.1999.0385p.xCitations: 32AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract 1 Recombinant wild-type or mutant human ρ1 GABA receptors were expressed in human embryonic kidney (HEK) 293 or monkey COS-7 cells and studied using the patch clamp technique. 2 Standard whole-cell recordings with 4 mM Mg-ATP in the patch pipette induced a time-dependent decrease in the GABA-activated current (IGABA) amplitude that was not the result of a decrease in GABA sensitivity. In contrast, IGABA remained stable when recordings were obtained using the perforated patch configuration or with standard whole-cell recording and no Mg-ATP in the patch pipette. 3 The inhibitors of serine/threonine protein kinases KN-62 (20 μM) or staurosporine (20 nM) prevented the time-dependent decrease in the amplitude of IGABA seen in the presence of ATP. Alkaline phosphatase (220 U ml−1), when added to the patch pipette in the absence of ATP, induced a transient potentiation of IGABA. Although the protein kinase C (PKC) activator 4β-phorbol 12-myristate, 13-acetate (PMA) did not reduce the amplitude of IGABA, inclusion of the catalytic domain of PKC in the recording pipette accelerated the time-dependent decrease in current amplitude. These data suggest that phosphorylation is involved in the regulation of the amplitude of IGABA. 4 Mutation of the three PKC consensus sequences of the ρ1 receptor had no significant effect on the decline in IGABA, indicating that direct phosphorylation of these putative sites on the ρ1 receptor does not underlie the time-dependent decrease in amplitude. 5 In COS-7 cells transfected with wild-type ρ1 receptors, the amplitude of IGABA had completely recovered to the original value when the same cells were repatched after 30-40 min, indicating that the decline in IGABA was a reversible process. 6 The inhibitor of actin filament formation cytochalasin B, when added to the patch pipette in the absence of ATP, induced a time-dependent inactivation suggesting that the actin cytoskeleton may play a role in the regulation of the amplitude. 7 Coincident with the decrease in the amplitude of IGABA, the cell capacitance significantly decreased in the presence of ATP in the patch pipette. This decrease in capacitance was not observed in the absence of Mg-ATP. The decrease in the membrane surface area suggests that receptor internalization could be a potential mechanism for the observed inactivation. 8 At 32 °C, compared with 22 °C, the rate and magnitude of the decline was increased dramatically. In contrast, at 16 °C, no significant change in IGABA was observed over the 20 min recording time. This marked temperature sensitivity is consistent with receptor internalization as a mechanism for the time-dependent decline in IGABA. 9 The specificity of the decrease in IGABA was assessed by coexpressing the voltage-dependent potassium channel Kv1.4 along with the ρ1 receptor in HEK293 cells. The amplitude of the potassium current (IKv1.4) exhibited very little decrement in comparison to IGABA suggesting that the putative GABA receptor internalization was not the consequence of a non-specific membrane retrieval. GABAC receptors, presumably consisting of rho subunits (Cutting et al. 1991; Wang et al. 1994), are a class of inhibitory GABA-activated channels in the central nervous system (Polenzani et al. 1991; Feigenspan et al. 1993; Qian & Dowling, 1994; Strata & Cherubini, 1994; Enz et al. 1996). Similar to GABAA receptors, they possess a high permeability to Cl−, but in contrast to GABAA channels, they are insensitive to bicuculline, barbiturates and benzodiazepines (Polenzani et al. 1991). The activity of GABAC receptors is regulated by extracellular agents, such as Zn2+, H+, Ca2+ (Wang et al. 1995; Kaneda et al. 1997), and also by intracellular factors, such as Ca2+, phosphatases and kinases (Feigenspan & Bormann, 1994b; Kusama et al. 1995). Protein phosphorylation is postulated to be an important physiological mechanism for regulating GABA-mediated synaptic inhibition (Moss et al. 1992; Moss & Smart, 1996). Neuronal GABAC channel currents demonstrate a Ca2+- and ATP-dependent decrease in amplitude during prolonged whole-cell recording (Feigenspan & Bormann, 1994b). In addition, the protein kinase C (PKC) activator 4β-phorbol 12-myristate, 13-acetate (PMA) decreased the amplitude of GABA-activated currents from homomeric ρ1 receptors expressed in Xenopus oocytes (Kusama et al. 1995; Chapell et al. 1998). Consensus sites for phosphorylation are located in the large intracellular loop between the putative third and fourth transmembrane domains of the ρ1 homomeric GABA receptor (Cutting et al. 1991), suggesting that direct phosphorylation of the ρ1 receptor could be a potential mechanism for regulation of the current amplitude. Alternatively, the modulation could be due to a PKC-dependent alteration in other proteins that interact with, and regulate, the ρ1 receptor. The present study was designed to distinguish between these and other possible regulatory mechanisms of the ρ1 receptor. ρ1 receptors were expressed in human embryonic kidney (HEK) 293 or monkey COS-7 cells and examined with whole-cell recording techniques. When ATP was included in the recording pipette, a time-dependent decrement in the amplitude of the GABA-activated current (IGABA) was observed. The decrement in amplitude was not observed in the absence of ATP or with perforated patch recordings. Although kinase inhibitors prevented the decline in IGABA, and the presence of the catalytic domain of PKC in the patch pipette hastened the decline, elimination of the three PKC consensus sites did not prevent the decrease indicating that direct phosphorylation of these particular residues is not required for the modulation. Experiments examining the actions of cytochalasin B, as well as those monitoring the cell capacitance throughout the recordings, suggest a phosphorylation-dependent receptor internalization via interaction with the cytoskeleton as a mechanism of the time-dependent inactivation of IGABA. METHODS Molecular biology The human ρ1 subunit was obtained via the polymerase chain reaction as described previously (Amin & Weiss, 1994) and subcloned into the pALTER-1 vector (Promega, Madison, WI, USA) for site-directed mutagenesis using Altered Sites (Promega). Mutagenesis was verified by cDNA sequencing and both the wild-type and mutant ρ1 receptors were subcloned into pCDNA3 (Invitrogen, San Diego, CA, USA). The human Kv1.4 clone was kindly provided by C. Garner (UAB, Birmingham, AL, USA) in the pCMV vector. Cell culture and transfection HEK293 cells (ATCC CRL 1573) or COS-7 cells (ATCC CRL 1651) were plated onto poly-L-lysine-coated glass coverslips (12 mm) placed in a 35 mm culture dish and maintained in Dulbecco's modified Eagle's medium-Ham's F12 (DMEM-F12; Gibco BRL, Gaithersburg, MD, USA) (HEK293) or DMEM (COS-7) supplemented with 10 % fetal bovine serum (Atlanta Biologicals, Norcross, GA, USA). The following day, transient transfections were performed using Lipofectamine (Gibco BRL) in the same media without serum or antibiotic according to the manufacturer's protocol. The ρ1-containing plasmid was cotransfected with the green fluorescent protein (GFP)-containing plasmid pGreenLantern (Gibco BRL) in order to enable visual identification of transfected cells. A total of 2 μg of cDNA was used per 35 mm plate; typically 1:1 for the ρ1:pGreenLantern plasmids. Cells were maintained in normal serum-containing medium and recordings were made 2-4 days post transfection. The HEK293 cell line that stably expressed ρ1 receptors was produced by carrying out a standard transfection as described above and performing cell passages for 1-2 months in the presence of 400 mg l−1 of G-418 (Gibco BRL). Cells were then plated at a low density and islands of cells were removed with a pipette after light trypsinization. These clonal cell lines were then tested for ρ1 receptor expression. Stable cells were maintained in 200 mg l−1 G-418. Electrophysiology Experiments were performed at room temperature (20-24°C) using the perforated patch and whole-cell recording techniques. Cells were visualized with an inverted microscope equipped with fluorescence. Currents were recorded using an Axopatch-1D amplifier (Axon Instruments, Foster City, CA, USA) and digitized (5-10 kHz) on a Macintosh computer using Pulse (Heka Electronik, Lambrecht, Germany). Analysis was carried out with Igor (Wavemetrics Inc., Lake Oswego, OR, USA). The external recording solution contained (mM): NaCl, 140; KCl, 3·5; glucose, 10; CaCl2, 2; Hepes, 10 (pH 7·2). The recording borosilicate glass pipettes had resistances of 2-4 MΩ when filled with the internal solution containing (mM): CsCl, 130; CaCl2, 0·25; EGTA, 1·1 (free Ca2+, ∼5 × 10−8 M); Hepes, 10 (pH 7·4). When indicated, 4 mM Mg-ATP, 220 U ml−1 alkaline phosphatase, 20 nM staurosporine, 20 μM KN-62, 0·4-4 nM of the catalytic domain of PKC, or 2 μg ml−1 cytochalasin B was added to the recording pipette. Staurosporine, KN-62 and cytochalasin B were dissolved in DMSO and diluted 1000-fold prior to use. The holding potential was -50 mV in all experiments. GABA (from 0·5 to 20 μM), dissolved in external solution, was applied to the cells through a double-barrel fast piezoelectric perfusion system. In some experiments, bicuculline (10 μM) was also added to the external solution. For the temperature experiments, the perfusion solution was warmed using a TC-344A heater controller (Warner Instruments, Hamden, CT, USA) or cooled by passing the inlet line through a cold water bath. In both cases, the temperature was monitored in the bath just beyond the target cell. For the experiments examining activation of PKC by PMA, HEK293 cells were switched to serum-free medium 24 h prior to the addition of PMA. PMA (0·5-0·8 μM) was introduced to the cells 1 h prior to recording. Perforated patch recordings were performed using amphotericin B (240 mg ml−1) in the pipette solution containing (mM): NaCl, 140; KCl, 3·5; glucose, 10; Hepes, 10 (pH 7·2). Amphotericin B was dissolved in DMSO and diluted 1000-fold for use. The tip of the recording pipette was dipped for ∼1 s in control solution (without amphotericin) before the pipette was backfilled with the amphotericin B solution. Access resistances typically stabilized within 3-15 min after the patch formation and reached 15-40 MΩ. The value of the access resistance was estimated using a 10 mV depolarizing pulse. Changes in the cell capacitance were approximated by integrating the capacitive current transients in response to this 10 mV voltage pulse (Lindau & Neher, 1988). The current amplitude and cell capacitance were normalized to the initial value (30-60 s from the begining of recording) in each of the experiments. All results are presented as means ±s.e.m. Data were compared statistically by Student's t test. Statistical significance was determined at the 5 % level. The experiments examining the temperature dependence of the decrease in IGABA and the effects of PKC in the patch pipette were carried out on a HEK293 cell line stably expressing ρ1 receptors. The properties of the receptors in the stable cell line were indistinguishable from those of the transiently expressed ρ1 receptors, but the expression level was less variable and the magnitude of the time-dependent decline in IGABA over time was slightly less than that observed in the transient transfections. Currents from Kv1.4 channels were examined by applying a 200 ms voltage step from -80 to +40 mV. The leak was determined by stepping the membrane potential from a holding potential of -40 mV to +40 mV. The plotted amplitude (IKv1.4) was the peak of the leak subtracted current trace. Drugs Cytochalasin B, staurosporine, KN-62, and alkaline phosphatase were obtained from RBI (Natick, MA, USA), bicuculline and amphotericin B were obtained from Sigma, and PMA and the catalytic domain of protein kinase C were obtained from Calbiochem (San Diego, CA, USA). RESULTS Basic properties of recombinant ρ1 receptors expressed in HEK293 cells Figure 1A shows typical GABA-activated whole-cell currents (IGABA) recorded at a range of membrane potentials from HEK293 cells expressing recombinant homomeric ρ1 GABA receptors. As previously demonstrated, ρ1 GABA-activated currents exhibited little desensitization with prolonged GABA application. With an IGABA greater than 2 nA, however, we typically observed a slow decay during continuous GABA application. This decay was associated with a slight shift in the reversal potential (not shown), suggesting a change in the intracellular Cl− concentration. Voltage ramps indicated that the current-voltage (I-V) relationship was linear (Fig. 1A; I-V plot). The 10-90 % rise times were 1132 ± 80 and 495 ± 41 ms (n= 5 cells) for currents activated by 10 and 20 μM GABA, respectively (not shown). The time constant of decay upon GABA removal (deactivation) was 9·4 ± 0·9 s (n= 23; Fig. 1B). Figure 1Open in figure viewerPowerPoint Properties of wild-type recombinant ρ1 receptors transiently expressed in HEK293 cells A, responses to GABA (10 μM, 20 s duration) applied through a fast perfusion system at the indicated holding potentials. The whole-cell current-voltage relationship (I-V), as determined with voltage ramps, was linear. B, decay of the current upon GABA removal (deactivation) was well described by a single exponential component with a time constant (τ) of 11 s. In 23 cells, the time constant of decay was 9·4 ± 0·9 s. C, whole-cell currents evoked at a holding potential of -50 mV with GABA (0·5, 1, 5 and 10 μM) application through a fast perfusion system (top) and normalized concentration-response curve for GABA-evoked currents (bottom). Hill, Hill coefficient. D, bicuculline (10 μM) did not block the response to GABA (10 μM, 20 s duration). Figure 1C shows currents activated by 0·5, 1, 5 and 10 μM GABA and the corresponding dose-response relationship. The continuous line is the best fit of the Hill equation to these data and revealed an EC50 for GABA (concentration required for half-maximal activation) of 0·90 ± 0·09 μM and a Hill coefficient of 3·0 ± 0·5 (n= 3). Recombinant homomeric ρ1 receptors are insensitive to the GABAA receptor antagonist bicuculline. Figure 1D demonstrates that 10 μM bicuculline had no effect on GABA-activated currents from HEK293 cells transfected with ρ1 receptors (n= 3), indicating the lack of endogenous GABAA currents that are sometimes observed in HEK293 cells (Ueno et al. 1996). Thus, the data in Fig. 1 demonstrate that the main properties of recombinant ρ1 receptors expressed in HEK293 cells are similar to those of recombinant ρ1 receptors expressed in oocytes (Cutting et al. 1991; Amin & Weiss, 1994) as well as those of GABAC receptors in neurons (Feigenspan & Bormann, 1994a; Qian & Dowling, 1994). Inactivation of ρ1 receptors during whole-cell recording Figure 2A shows GABA-activated currents measured with standard whole-cell recording techniques during a 25 min recording period with 4 mM Mg-ATP in the patch pipette. Figure 2C shows a plot of the mean amplitude of IGABA normalized to that at the initial application of GABA. Note that the amplitude declined to 0·56 ± 0·09 (n= 6) of the initial value during the first 15 min of recording and then stabilized. The ratios of the current amplitudes with 1 and 5 μM GABA after 1 and 15 min of whole-cell recording were 0·74 ± 0·14 and 0·71 ± 0·10, respectively (Fig. 2B, n= 3). Thus, the decline in the amplitude of IGABA was not the result of a decrease in agonist sensitivity. Figure 2Open in figure viewerPowerPoint ATP in the recording pipette induced inactivation of GABA-activated currents in HEK293 cells expressing ρ1 receptors during whole-cell recording A, currents evoked by GABA (20 μM) application through a fast perfusion system decreased by ≈50 % during 15 min of whole-cell recording in the presence of ATP (4 mM) in the patch pipette. In B, the traces represent the currents activated by 1 and 5 μM GABA at different times during the recording in the presence of ATP in the patch pipette. The ratios of current amplitudes activated by 1 and 5 μM GABA were 0·62 and 0·68 before and after inactivation of the current amplitude, respectively. C, mean normalized GABA-activated current amplitude in the presence of ATP during prolonged recording. In six cells, the amplitude fell to 0·56 ± 0·09 during 15 min of recording. Wild-type ρ1 GABA-activated currents are stable with perforated patch recording or in the absence of Mg-ATP in the patch pipette HEK293 cells expressing homomeric ρ1 receptors were voltage clamped using the perforated patch technique and 10 μM GABA was applied at 5 min intervals. As illustrated in Fig. 3A, the amplitude of IGABA was stable during prolonged recording (the normalized amplitude was 1·05 ± 0·04 after 25 min of recording; n= 5). Figure 3B shows a plot of the amplitude of IGABA (filled circles) and the access resistance (Raccess; open circles), both of which remained stable over this time period. Thus, the amplitude of IGABA in HEK293 cells transfected with ρ1 receptors and recorded using the perforated patch technique remained relatively constant during long term recording. Figure 3Open in figure viewerPowerPoint The amplitude of the GABA-activated current remained stable during perforated patch recording or without ATP in the recording pipette A, currents evoked by GABA application through a fast perfusion system to cells expressing wild-type ρ1 receptors at different times during perforated patch recording. B, mean GABA-activated current amplitude (•) and access resistance (○) during prolonged recording. Note that the amplitude and the access resistance remained stable. The normalized values were 1·05 ± 0·04 and 0·98 ± 0·12 (n= 5) after 25 min of recording, respectively. C, currents evoked by GABA application through a fast perfusion system to cells expressing ρ1 receptors at different times during standard whole-cell recording and without ATP in the recording pipette. D, mean GABA-activated current amplitude in the absence of ATP in the recording pipette (•). ○indicates the mean amplitude in the presence of ATP (Fig. 2C) replotted for comparison. Note that, in the absence of ATP, the current amplitude remained stable for the first 20 min of recording. Similarly, without the addition of Mg-ATP to the patch pipette (Fig. 3C), the current amplitude did not change significantly during 15 min of recording (0·97 ± 0·09 of the initial value, n= 7; Fig. 3D, filled circles). In most cells, a decrease in the amplitude began to develop after 20 min of recording. The open circles in Fig. 3D are the data in the presence of ATP replotted for comparison. The requirement for ATP in this time-dependent inactivation of IGABA suggests phosphorylation as a possible mechanism. Serine/threonine-dependent phosphorylation is involved in the regulation of ρ1 receptors To gain further insight into the mechanism of this ATP-dependent inactivation of IGABA, we examined the effects of KN-62 (20 μM), an inhibitor of Ca2+-calmodulin (CaM)-dependent protein kinase, and staurosporine (20 nM), an inhibitor of PKC. As shown in Fig. 4A and B and the plot of IGABA over time (Fig. 4D), these protein kinase inhibitors prevented the ATP-dependent decrease in the amplitude of IGABA. The normalized amplitudes were 0·97 ± 0·1 (n= 5) and 0·96 ± 0·16 (n= 5) after 15 min of recording for KN-62 and staurosporine, respectively. Alkaline phosphatase (220 U ml−1), added to the recording pipette without ATP, induced an initial (although transient and highly variable) potentiation of IGABA during the first 10 min of recording (Fig. 4C and D). The data in Fig. 4 implicate PKC- and Ca2+-CaM-dependent phosphorylation as candidate pathways for the ATP-dependent inactivation of homomeric ρ1 GABA receptors. Figure 4Open in figure viewerPowerPoint PKC and Ca2+-CaM-dependent phosphorylation may be involved in the modulation of the amplitude of IGABA In A-C, traces represent currents evoked by GABA (20 μM) application through a fast perfusion system to cells expressing wild-type ρ1 receptors in the presence of KN-62 (20 μM; A), staurosporine (20 nM; B) and alkaline phosphatase (220 U ml−1; C). Note that, in all cases, the GABA-evoked current did not decrease during 25 min of recording. D, mean normalized GABA-activated current amplitudes in the presence of staurosporine (▪), KN-62 (○) or alkaline phosphatase (•) in the recording pipette. In order to examine whether the PKC pathway was involved in the ATP-dependent inactivation, we compared the amplitude of IGABA with and without a 1 h incubation in the presence of the PKC activator PMA (0·5-0·8 μM). The IGABA amplitudes were 796 ± 180 pA (n= 6) and 747 ± 70 pA (n= 6), with and without PMA, respectively. We also tested the effects of including the catalytic domain of PKC (0·4-4 nM) in the patch pipette on the ATP-dependent decline in IGABA. Figure 5A and B shows examples of IGABA throughout a 20 min recording period in the absence and presence, respectively, of 1 nM PKC. The mean peak values of IGABA from five cells with and without PKC in the recording pipette are plotted in Fig. 5C. The presence of PKC increased the ATP-dependent decrease in IGABA. The amplitude of IGABA decreased to 0·77 ± 0·05 (n= 5) of the original value in comparison with a decrease to 0·46 ± 0·08 (n= 5) in the presence of PKC. The experiments in which the HEK293 cells were incubated in PMA suggest that either PKC was already maximally stimulated or the PKC pathway may not be directly involved in the ATP-dependent decline. Nevertheless, the enhanced decline in the presence of PKC supports the conclusion that a phosphorylation-dependent mechanism could underlie the time-dependent decrease in IGABA. Figure 5Open in figure viewerPowerPoint Inclusion of the catalytic domain of PKC in the patch pipette enhanced the decline in IGABA A, GABA-activated currents in a HEK293 cell stably expressing ρ1 receptors over a 20 min recording period. B, same as in A, but 1 nM of the catalytic domain of PKC was included in the recording pipette. Note the enhanced decline in IGABA in the presence of PKC. C, mean normalized GABA-activated current amplitudes in the absence and presence of PKC in the patch pipette. After 20 min IGABA decreased to 0·77 ± 0·05 (n= 5) of its original value while, in the presence of PKC, IGABA decreased to 0·46 ± 0·08 (n= 5) of its original value. Phosphorylation of the PKC consensus sites S410, S419 and S426 is not required for the IGABA inactivation The ρ1 receptor has three serine residues in the intracellular loop between the putative third and fourth transmembrane domains that are potential PKC phosphorylation sites (Cutting et al. 1991). One possible mechanism for the ATP-dependent decline in IGABA might involve direct phosphorylation of the ρ1 subunit. To evaluate whether phosphorylation of these sites may be directly involved in the ATP-dependent inactivation of GABA receptor function, we examined recombinant ρ1 receptors in which all three serines were mutated to alanine (S/410-419-426/A). Figure 6A shows GABA-activated currents from HEK293 cells transfected with the triple mutant. Note that, similar to the wild-type receptor (Fig. 2A), the current continuously decreased during 20 min of recording. The filled circles in Fig. 6B show IGABA normalized to the value at the initial GABA application. The dashed line and open symbols are the wild-type data (Fig. 2C) replotted for comparison. The current amplitude of the mutant decreased to 0·59 ± 0·09 (n= 7) of the initial value during 25 min of recording. These results indicate that phosphorylation of the serine residues S410, S419 or S426 is not necessary for the ATP-dependent decrease in the amplitude of IGABA. Figure 6Open in figure viewerPowerPoint Mutation of the PKC consensus sites on the ρ1 receptor did not prevent the inactivation of IGABA A, currents evoked by GABA (10 μM) applications to HEK293 cells expressing the triple mutant ρ1 receptors (S/410-419-426/A) in the presence of ATP (4 mM) in the intracellular solution during whole-cell recording. Note that the current amplitude decreased ≈33 % during 20 min of whole-cell recording. B, mean normalized GABA-activated current amplitude of the triple mutant ρ1 receptors in the presence of ATP in the intracellular solution (•). Note the significant decrease (0·59 ± 0·09, n= 7, P < 0·05) in the current amplitude during 25 min of recording. ○, current amplitude of the wild-type ρ1 receptor replotted for comparison. The time-dependent inactivation of ρ1 receptors is a reversible process Next, we set out to determine whether, after declining, the amplitude of IGABA could recover back to its original value. Reversibility would place some constraints on potential mechanisms. These particular experiments were carried out in COS-7 cells, rather than in HEK293 cells, since (at least in our hands) the COS-7 cells were much more easily repatched. In the presence of ATP, the amplitude of the GABA-induced current continuously dropped to 0·60 ± 0·06 (n= 7) of the initial value during 30 min of recording (first four current traces in Fig. 7A and the filled circles in Fig. 7B). Thus, the features of the time-dependent inactivation of IGABA in COS-7 cells were qualitatively similar to those observed in HEK293 cells. Figure 7Open in figure viewerPowerPoint The ATP-dependent inactivation of IGABA is reversible in COS-7 cells transfected with wild-type ρ1 receptors A, traces are GABA-activated currents during whole-cell recording. The trace on the right is the GABA-activated current when the same cell was repatched 30 min after the initial recording was terminated. Note that during 28 min of initial recording the amplitude decreased ≈38 % and then fully recovered to its original value. B, the normalized GABA-activated current amplitude continuously decreased during prolonged recording in the presence of ATP in the intracellular solution. Upon repatching (after 30 min) the amplitude had recovered to its original value and then decayed again. C, mean GABA-evoked current amplitudes normalized to the amplitude at the first minute of initial recording. Note that upon repatching after 25-35 min from the end of the initial recording, the current amplitude had returned to 0·94 ± 0·07 of the original value. We then repatched the same cell after 30 min from the end of the initial whole-cell recording (right-hand trace in Fig. 7A). In this experiment, the current amplitude dropped to almost half during 38 min of initial recording and recovered to nearly its original value after repatching, before declining again (Fig. 7B, open circles). Figure 7C shows the mean results from four such experiments. IGABA decreased to 0·55 ± 0·06 (n= 4) during 30-40 min of initial recording and recovered to 0·94 ± 0·07 (n= 4) of the initial value after repatching. A recovery in the amplitude was not observed if we maintained the initial recording for 15-20 min after inactivation, but without GABA application (n= 2; data not shown). These data eliminate irreversible mechanisms such as receptor degradation for the decline in the amplitude of IGABA. The actin cytoskeleton may be involved in the regulation of ρ1 GABA receptors The cytoskeleton has been show