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J Physiol (2003), 550.3, pp. 693-706
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.042119
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ABSTRACT |
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Cerebellar granule (CG) neurons express a G protein-gated K+ current (GIRK) that is involved in the neurotransmitter regulation of the excitatory input to the Purkinje fibres of the cerebellum. Here, we characterized the single-channel behaviour of GIRK in CG neurons, and examined the effects of several known modulators of GIRK and their putative physiological roles. Whole-cell GIRKs were activated by baclofen, a GABAB receptor agonist. In cell-attached patches, baclofen activated GIRK with a single-channel conductance of 34 pS and a mean open time of 0.5 ms. In inside-out patches, application of GTPS to the cytoplasmic side activated GIRK with similar kinetic properties. Addition of 2 mM ATP resulted in a marked increase in GIRK activity and induced longer-lived openings with a mean open time of 2.3 ms (ATP-dependent gating). Brain cytosolic fraction or free fatty acids inhibited this effect of ATP, and this was reversed by addition of purified recombinant brain fatty acid binding protein. Applying phosphatidylinositol 4,5-bisphosphate (PIP2) to inside-out patches in place of ATP also increased GIRK activity; however, only an increase in the frequency of opening was observed. The stimulatory effect of PIP2 on GIRK activity was not inhibited by the cytosolic fraction. Following maximal activation by PIP2, ATP caused an additional 2.2-fold increase in GIRK activity. These results show that GIRKs in CG neurons are regulated by positive and negative modulators that affect frequency as well as open time duration. The net effect is that the ligand-activated GIRK is in the 'low activity' state associated with short-lived openings, mainly due to strong action of the cytosolic inhibitor of ATP-dependent gating. Our results also show that intracellular ATP modulates GIRK via pathways different from that of PIP2 in CG neurons.
(Received 21 February 2003; accepted after revision 7 May 2003; first published online 13 June 2003)
Corresponding author D. Kim: Department of Physiology and Biophysics, Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064, USA. Email: donghee.kim{at}finchcms.edu
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INTRODUCTION |
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The G protein-gated K+ channel (GIRK) is normally activated by ligands that bind receptors that are coupled to Gi/o proteins (Kurachi, 1995; Stanfield et al. 2002). Several recent studies have shown that the 
subunit of Gi/o protein binds to the cytoplasmic domain of GIRK and causes channel activation (Dascal et al. 1995; Huang et al. 1995; Krapivinsky et al. 1995b; Doupnik et al. 1996; Kofuji et al. 1996; Huang et al. 1997). In isolated membrane patches, GIRK activity has been shown to be modulated by various cellular molecules and factors that include MgATP, phosphatidylinositol 4,5-bisphosphate (PIP2), Na+ and free fatty acids (Kim, 1991; Sui et al. 1996; Huang et al. 1998; Ho & Murrell-Lagnado, 1999; Kim & Pleumsamran, 2000). Among these modulators, PIP2 has received much attention as it was initially reported to be critical for GIRK activation by (Huang et al. 1998). Subsequent studies showed that the presence of PIP2 in the membrane is necessary for GIRK activation not only by but also by Na+ (Sui et al. 1996).
One modulatory factor that has been equated to PIP2 is intracellular ATP. In atrial membrane patches, application of alone causes a rapid activation of GIRK and further addition of ATP in the presence of Mg2+ produces a marked increase in GIRK current (Kim, 1991; Sui et al. 1996). The ATP-induced stimulation of GIRK activity is thought to be due to an increase in the concentration of PIP2 in the membrane generated via lipid kinases (Huang et al. 1998; Sui et al. 1998). As the intracellular concentration of ATP is normally high at 4-5 mM in intact cells, it seems likely that the concentration of PIP2 in the membrane is maintained near an optimal level and undergoes only transient changes during activation of phospholipase C via receptor ligands. Other physiological factors that regulate the activity of lipid kinases and phosphatases in the cell probably also determine the concentration of PIP2. In addition to ATP and PIP2, a potent inhibitory factor was recently found to be present in the brain and heart cytoplasm and to modulate the activity and the gating mode of atrial GIRK (Kim & Pleumsamran, 2000). This unidentified factor was able to fully block the stimulatory effect of ATP on GIRK in atrial cell membrane patches. Analysis of the cytosolic preparation suggested that the inhibitory factor was a lipid rather than a protein. Direct test of various lipids on GIRK function showed that free fatty acids were able to produce effects similar to the cytosolic preparation that contained the inhibitory factor. Nevertheless, strong evidence in support of free fatty acids as the endogenous intracellular negative modulator of GIRK has been lacking. Whatever the true identity of the inhibitor of GIRK, the physiological activation of GIRK by an agonist that occurs in intact cells will be the net sum of the effects produced by various positive and negative modulators that exist in the cell during agonist-induced activation.
The properties of GIRK have been studied mostly in atrial cells and in cells expressing cloned GIRK subunits. Only a few studies have described the behaviour of native GIRK in neuronal cells (Oh et al. 1995; Bajic et al. 2002; Stanfield et al. 2002), and the roles of various modulators of GIRK have yet to be studied in neurons. Furthermore, GIRK with different rectifying properties have been described in neurons from different brain regions, suggesting that there may be more than a single type of GIRK channel. These GIRK channels may possess different modulatory properties, possibly due to different subunit compositions (Sodickson & Bean, 1996; Stanfield et al. 2002). Therefore, we examined the properties and modulation of GIRK in neurons and compare them with those obtained in atrial cells. In this study, we investigated the properties and modulation of GIRK in cerebellar granule (CG) neurons that form synapses with Purkinje cells and provide a major excitatory input via parallel fibres in the cerebellum. This excitatory input can be modulated by neurotransmitters that act on receptors that are coupled to GIRK in CG neurons. An inhibitory neurotransmitter that can activate GIRK in CG neurons is -aminobutyric acid (GABA) that acts on the metabotropic GABAB receptor (Bowery, 1993; Misgeld et al. 1995). Other signalling molecules such as anandamide, an endogenous cannabinoid, and acetylcholine may also activate GIRK via their specific receptors in CG neurons (Jung et al. 1997; Ho et al. 1999; McAllister et al. 1999). Whole-cell GIRK currents activated by GABAB receptor agonists have been described in previous studies (Kofuji et al. 1996; Surmeier et al. 1996; Slesinger et al. 1997). Our study shows the first detailed characterization of the G protein-gated K+ channel and its regulation by ATP, PIP2 and cytosolic inhibitors in CG neurons at the single-channel level.
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METHODS |
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Cerebellar granule neuron culture and isolation
All animals were used in accordance with the Guide for the Care and Use of Laboratory Animals (DHEW Publication No. NIH85-23). The cerebellum was isolated from decapitated postnatal day (P)6-P8 rat pups (Sprague-Dawley) and washed with oxygenated physiological buffer solution at 4 °C. The cerebellar cortex was cut into thin sections and incubated for 15 min in a solution containing papain (12 units ml-1; Worthington, Lakewood, NJ, USA), albumin (0.2 mg ml-1) and DL-cysteine (0.2 mg ml-1). After digestion, the tissue was washed twice with phosphate-buffered saline (PBS) and resuspended in a solution containing DNase I (1000 Kunitz ml-1; Worthington). After gentle trituration of the solution using a fire-polished glass pipette, the suspended cells were gently passed through a 3 ml, 25 gauge syringe. The suspension was layered on top of sterilized fetal bovine serum and centrifuged at 100 g for 10 min. The pellet was resuspended in plating medium that contained Neurobasal Medium supplemented with B-27 (10 µl ml-1; Life Technologies, Rockville, MD, USA), glutamic acid (2.5 mM), glutamine (20 mM), gentamicin (50 µg ml-1) and fungizone (2.5 µg ml-1). The cells were plated on glass coverslips coated with poly-L-lysine at a density of 1 
105 cells cm-2. After a 24 h period for cell attachment, the medium was changed every 3 days with new plating medium containing B-27 (20 µl ml-1), glutamine, gentamicin and fungizone in Neurobasal Medium. Cells were kept at 37 °C in a humidified incubator gassed with 95 % air : 5 % CO2 mixture.
Purification of fatty acid binding protein
Rat brain fatty acid binding protein (FABP) cDNA was a gift from Dr B. Popko (University of North Carolina, USA). cDNA containing sequences that encode FABP-glutathione-S-transferase (FABP-GST) was constructed and subcloned into the expression vector pGEX-2T that was transformed into BL21 E coli. Expression of FABP (optical density (OD)600 = 0.8) was induced with 0.1 mM isopropyl-1-thio--D-galactoside (IPTG) for 4 h in the shaking incubator at 37 °C. The cells were sedimented by centrifugation at 3300 g for 20 min. Supernatant was removed and the cell pellet was washed once with cold PBS and then resuspended in PBS. The resuspended pellet was disrupted using a sonicator followed by centrifugation at 9300 g for 45 min. The supernatant (cytosolic extract) was filtered through a 0.45 µm filter and loaded onto a column packed with Glutathione Sepharose 4B (Pharmacia Biotech, Sweden). After three washes, each with 5 bed volumes of PBS, proteins bound to the beads were treated with thrombin (125 units ml-1, without bovine serum albumin; Sigma, St Louis, MO, USA) diluted in thrombin cleavage buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2) to cleave off FABP from GST. The column treated with thrombin was incubated overnight with gentle agitation at room temperature. After incubation, thrombin cleavage buffer was loaded onto the column to elute free FABP. GST bound to beads was eluted using 5 bed volumes of glutathione elution buffer (10 mM glutathione in 50 mM Tris-HCl, pH 8.0). Three fractions (cytosolic extract (lane 1), eluted buffer containing FABP (lane 2) and eluted buffer containing GST (lane 3)) were prepared and proteins were separated on a 12 % gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie brilliant blue R250 (Sigma), as shown in Fig. 1.
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Figure 1. Separation of proteins by SDS-PAGE Lane 1, cytosolic extract of bacteria after 4 h of induction with isopropyl-1-thio- | ||
Preparation of cytosol
Rats were quickly decapitated and the brain was quickly removed. Whole brains (~5 g) were homogenized for ~30 s in ice using a polytron in 5 ml Hepes buffer (containing (mM): 50 Hepes, 5 EDTA (pH 7.4), 0.5 dithiothreitol and 0.1 phenylmethylsulfonyl fluoride). Homogenized tissue was then centrifuged at 3300 g for 20 min at 4 °C (Beckman Model J2-21M, rotor JA-21). The supernatant was collected and mixed with the same volume of cytoplasmic extract buffer containing (mM): 140 KCl, 3.0 MgCl2 and 30 Hepes (pH 7.9). This mixture was centrifuged at 100 000 g for 1 h using a Beckman L-60 ultracentrifuge (rotor 70-TI). The supernatant was placed in tubing for dialysis (Spectrum; 10 000 molecular weight cutoff). Dialysis was carried out in 4 l of solution containing (mM): 140 KCl, 2 MgCl2, 5 EGTA, 0.5 dithiothreitol, 0.1 phenylmethylsulfonyl fluoride (PMSF), 10 Hepes (pH 7.2) with leupeptin (1 mg ml-1) and pepstatin A (1 mg ml-1) for 12 h at 4 °C. This cytosolic fraction (20 mg ml-1 protein) was stored at -70 °C. Protein concentration was determined by Bradford assay (Sigma).
Electrophysiological studies
Electrophysiological recording was performed using a patch clamp amplifier (Axopatch 200, Axon Instruments, Union City, CA, USA). All recordings were performed at room temperature (~24 °C). Single-channel currents were digitized with a digital data recorder (VR10, Instrutech, Great Neck, NY, USA), and stored on videotape. The recorded signal was filtered at 5 kHz using an 8-pole Bessel filter (-3 dB; Frequency Devices, Haverhill, MA, USA) and transferred to a computer (Dell) using the Digidata 1200 interface (Axon Instruments) at a sampling rate of 20 kHz. Threshold detection of channel openings was set at 50 %. Whole-cell currents were recorded after cancelling the capacitive transients. Whole-cell and single-channel currents were analysed with the pCLAMP program (Version 7). Data were analysed to obtain a duration histogram, an amplitude histogram and an NPo value. N is the number of channels in the patch and Po is the probability of a channel being open. NPo was determined from ~1 min of current recording. Expanded single-channel current tracings shown in the figures were filtered at 2 kHz. In experiments using excised patches, pipette and bath solutions contained 140 mM KCl, 1 mM MgCl2, 5 mM EGTA and 10 mM Hepes. The pH was adjusted to 7.3 using KOH. In whole-cell recordings, bath solution contained 135 mM NaCl, 3 mM KCl, 0.5 mM CaCl2, 1 mM MgCl2 and 10 mM Hepes. The pH was adjusted to 7.3 using NaOH. Free fatty acids and PIP2 were dissolved by sonication for 5 min (Heat Systems-Ultrasonics, Inc., W-380, Farmingdale, NY, USA) in bath recording solution at the desired concentration. All other chemicals were purchased from Sigma. PIP2 (L-
-phosphatidyl-D-myo-inositol-4,5-bisphosphate) was purchased from Sigma and Biomol. For statistics, Student's t test was used with P < 0.05 as a criterion for significance. Data are represented as means ± standard deviation.
Single-channel analysis
As nearly every patch contained multiple openings, a maximum likelihood algorithm was used to determine single-channel kinetic parameters from idealized patch clamp data (Qin et al. 1996). This method was used on data containing multiple channel openings (up to three levels) and estimated all transition rates between states after correction of missed events. Detailed description of this analysis can be found elsewhere and is therefore not given here (Qin et al. 1996). The best kinetic scheme that describes GIRK consists of three closed states and two open states, as shown in Scheme 1 (Nemec et al. 1999; Kim & Pleumsamran, 2000). We used this model to obtain rate constants between open and closed states. After rate constants were determined from ~6000 openings, open time constants were calculated as the value equal to 1 divided by the transition rate leading away from each open state. A kinetic scheme with a single open state (shown by dotted lines) was used to obtain the mean open time when the two open time constants were within 20 % of each other. Also, we made sure that the maximum logarithmic likelihood value for the fit for the single open state scheme was higher than the value obtained for the scheme with two open states, as described previously for GIRK (Kim & Bang, 1999; Kim & Pleumsamran, 2000).
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RESULTS |
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Activation of whole-cell GIRK current by baclofen
In CG neurons, GABA acts on GABAB receptors coupled to Gi/o protein to activate GIRK. We used baclofen, a specific GABAB agonist, to activate GIRK in CG neurons. In the whole-cell configuration, membrane potential was held at -60 mV to record an inward current in symmetrical 140 mM KCl solution. Extracellular application of 100 µM baclofen evoked a rapid activation of an inward current and washout of the drug decreased the current back to the baseline (Fig. 2A). The degree of GIRK activation varied markedly among CG neurons in culture. In 36 neurons (3 days in culture) with similar cell sizes as judged by cell capacitance (2.5-3.5 pF), we recorded peak currents ranging from 0 to 300 pA. The graph in Fig. 2A shows the distribution of cells according to the current magnitude activated by baclofen. Approximately half of the cells studied showed peak currents that were less than 30 pA pF-1. Baclofen failed to elicit any current in six cells. These results show that cultured rat CG neurons possess GIRK that can be activated via GABAB receptors, and suggest that the level of GIRK expressed in the membrane and/or the efficiency of signal coupling varies markedly among these neurons.
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Figure 2. Whole-cell currents activated by baclofen in cerebellar granule (CG) neurons A, cell membrane potential was held at -60 mV and baclofen (100 µM) was applied to the bath solution to activate GIRK current. Dashed lines indicate the zero current. The maximum current activated by baclofen was determined from each cell, and normalized to the cell size. Cells were divided into three groups, as shown. Inset, a photomicrograph of cerebellar granular cells in culture. B, baclofen was applied first to activate GIRK. After washing off baclofen, anandamide (10 µM) and WIN 55212-2 (10 µM) were applied sequentially. Baclofen was applied again at the end to check for activation of GIRK. A tracing typical of those observed in six experiments is shown. | ||
Earlier studies have reported that cannabinoid receptor (CB1) is expressed in rat CG neurons and that CB receptor agonists reduce cAMP concentration (Pacheco et al. 1993). In oocytes and COS-7 cells expressing CB receptors and GIRK, CB receptor agonists were found to activate GIRK (Jung et al. 1997; Ho et al. 1999). We tested whether CB receptor agonists activate GIRK in CG neurons by applying anandamide (10 µM) and WIN55212-2 (10 µM) to the perfusion solution. As shown in Fig. 2B, both agonists failed to affect the whole-cell current (n = 6 each). In the same cells, baclofen produced large GIRK currents, suggesting that the coupling of GIRK to G proteins associated with CB receptors may be lacking in our CG neurons early in culture (2-5 days). Methanandamide (10 µM), an analogue of anandamide that is resistant to degradation, also failed to activate GIRK that was subsequently activated by baclofen in the same cell (n = 4).
Single-channel properties of GIRK in CG neurons
CG neurons possess a large background K+ current that is sensitive to muscarinic receptor-mediated inhibition (Watkins & Mathie, 1996; Millar et al. 2000). In the majority of cell-attached patches formed from CG neurons in culture, openings of several background-like K+ channels were observed, as described earlier (Han et al. 2002). The background K+ channels consisted of tandem-pore K+ channels (TASK-1, TASK-3, TREK-2) and a pHo-sensing K+ channel whose gene has not yet been identified (Han et al. 2002). Although GIRK was not open at rest in cell-attached patches, its activation could be recorded in inside-out patches following application of GTPS. To study GIRK in isolation, we used CG neurons within 3 days after culture because of the lower expression of background K+ channels at early (1-3 days) growth stages than at later stages (4-10 days; Han, 2002). Even during the early growth stage, only ~10 % of patches could be used since most of the patches contained one or more of the background K+ channels. At later stages of growth, it was nearly impossible to study GIRK as nearly every patch showed high levels of background K+ channel activity.
Figure 3 shows a cell-attached patch with 100 µM baclofen in the pipette (tracing a) showing only single-channel openings of GIRK. GIRK activity in cell-attached patches studied was always low with an open probability (Po) less than 0.03 (0.022 ± 0.007; n = 6). Formation of inside-out patch resulted in a decrease in GIRK activity such that only one or two openings could be seen every few seconds (tracing b; Po < 0.001). Although not shown, applying 100 µM GTP to the bath solution resulted in an increase in GIRK activity (Po, 0.016 ± 0.006; n = 6), as expected. Applying 10 µM GTPS to the cytoplasmic side of the membrane produced a much greater (> 5-fold) activation of GIRK compared with that observed in cell-attached patches with baclofen (tracing c). The large effect of GTPS is presumably due to the irreversible accumulation of from all Gi/o proteins coupled to different receptors in the cell. Figure 3B shows current tracings at different holding potentials and illustrates the strongly inward rectifying property of GIRK in CG neurons. Duration and amplitude histograms obtained from channel openings at -80 mV are shown in Fig. 3C and D. The channels activated by GTPS alone in inside-out patches could be fitted by a single exponential function with a mean open time of 0.5 ± 0.1 ms (n = 5), identical to the channels activated by baclofen in cell-attached patches (0.5 ± 0.1 ms; n = 5). Amplitude histograms determined from channel openings at various membrane potentials were used to plot the current-voltage relationship shown in Fig. 3E. The single-channel conductance at -80 mV was 34 ± 2 pS. In keeping with the whole-cell current data, anandamide and WIN55212-2 (10 µM) added to the pipette solution failed to activate GIRK in cell-attached patches (n = 6 each). These results show that GIRK in CG neurons can be identified at the single-channel level, albeit with some difficulty caused by the abundance of background K+ channels, and that it has properties of GIRK1/2 heteromer, but not that of GIRK2 (see Discussion).
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Figure 3. Single-channel recording of GIRK in CG neurons A, a cell-attached patch was formed with baclofen in the pipette. After forming an inside-out patch, GTP | ||
Modulation of GIRK by intracellular factors
Intracellular ATP has been shown to support GIRK activity as well as to activate the channel (Lesage et al. 1995; Huang et al. 1998). We examined whether and how ATP modulates the native GIRK in CG neurons. Inside-out patches were formed and GTPS applied to activate GIRK. GIRK activated with GTPS showed only short-lived openings that could be described by a single open state, with a mean open time of 0.5 ± 0.1 ms. Addition of ATP (2 mM) to the perfusion solution, with free [Mg2+] kept constant at 1.0 mM, produced a marked increase in GIRK activity that was sustained. We show two representative tracings to illustrate the stimulatory effect of ATP on GIRK activity (Fig. 4A and B). The current tracing in Fig. 4A was observed often but could not be analysed for open lifetimes due to too many multiple openings. To determine the effect of ATP on open lifetimes, we used current tracings from patches with lower GIRK activity, as shown in Fig. 4B. The increase in activity produced by ATP was associated with long-lived openings, as determined from the analysis of the open time duration histograms (Fig. 4C). Single-channel analysis using the QuB program also indicated that in the presence of ATP, GIRK openings could be described using a model having two open states (Fig. 4E). We refer to this induction of longer-lived openings by ATP as 'ATP-dependent gating'. The second open state that was induced by ATP contributed 70 ± 12 % to total GIRK activity (n = 5). Such ATP-dependent gating was never observed in cell-attached patches of CG neurons. Therefore, rat brain cytosol (1 mg ml-1 protein) was prepared and applied to the patches in the presence of ATP. Upon addition of the cytosol, GIRK activity decreased to close to the pre-ATP level, and long-lived openings were no longer observed. Thus, the behaviour of GIRK in CG neurons was similar to that of atrial GIRK with respect to effects of ATP and the cytosol (Pleumsamran et al. 1998). These results show that GIRK activity observed in cell-attached patches of CG neurons is very low due to the potent inhibitory action of certain factors in the cytosol.
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Figure 4. Modulation of G protein-gated K+ current (GIRK) by ATP, cytosol and arachidonic acid (AA) A, inside-out patches containing no background K+ channels were chosen and GIRK was activated with GTP | ||
Earlier studies have shown that unsaturated free fatty acids inhibit the ATP-dependent gating of GIRK in atrial cells (Kim & Pleumsamran, 2000). Arachidonic acid (2 µM) was therefore applied to the patch in which GIRK was already activated fully by GTPS and ATP. Addition of arachidonic acid resulted in a marked reduction in GIRK activity and restored the gating kinetics back to that observed before ATP was applied (Fig. 4B). The effect of arachidonic acid was fully reversible and the ATP-dependent gating returned several minutes after washout of arachidonic acid as long as ATP was present. Two other free fatty acids (oleic and linoleic acids) were found to produce effects not statistically different from that of arachidonic acid on GIRK function in CG neurons (P > 0.05; n = 3). A summary of the results of these experiments is shown in Fig. 4D and E.
Effect of FABP on GIRK function
The finding that free fatty acids produce effects very similar to that of the cytosol suggested that intracellular free fatty acids may be the inhibitory component of the cytosol that blocks the ATP-mediated stimulation of GIRK activity. If this is true, a selective removal of free fatty acids from the cytosol should restore the ATP effect and produce a marked increase in GIRK activity, as well as show the ATP-dependent gating. To selectively remove free fatty acids, we prepared recombinant brain FABP in E. coli as a GST-FABP fusion protein. GST was cleaved off with thrombin, leaving free FABP. FABP stock (100 µM) was diluted to a final concentration of 5 µM in test buffer solution and applied to inside-out patches. Figure 5A shows an inside-out patch in which ATP produced a dramatic increase in GIRK activity. After completely inhibiting this effect of ATP by adding the cytosol, FABP was applied to the perfusion solution in the presence of the cytosol. GIRK activity gradually increased to a level close to that observed with GTPS and ATP (Fig. 5A). Washout of perfusion solution containing FABP and adding new cytosol quickly decreased GIRK activity. Thrombin itself or boiled FABP failed to affect GIRK activity. FABP itself had no significant effect on GTPS-activated GIRK activity (P > 0.05; n = 4).
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Figure 5. Reversal of the cytosol-induced inhibition of GIRK activity by FABP A, inside-out patches were obtained and GIRK activated with GTP | ||
In the second set of experiments, arachidonic acid was applied to inside-out patches to inhibit ATP-induced increase in GIRK activity. GIRK openings showed only short-lived openings after arachidonic acid treatment (tracing c, Fig. 5B). After FABP application, GIRK openings became more consistent with ATP-dependent gating, showing both short- and long-lived openings (tracing d and bar graphs). The most plausible explanation for these results is that FABP has selectively removed free fatty acids from the cytosol (Fig. 5A) and arachidonic acid (Fig. 5B) away from the modulatory sites of GIRK, allowing ATP-dependent gating to occur. These results provide additional evidence that cytoplasmic free fatty acids serve the endogenous inhibitor of GIRK and are critical in keeping GIRK in the 'low Po' state at rest, despite the strong positive modulation by ATP.
Modulation of GIRK by PIP2 in CG neurons
PIP2 is now well known to be an important component of signalling in GIRK function and has been suggested to mediate the effect of ATP (Huang et al. 1998; Sui et al. 1998). Therefore, we examined the direct effect of PIP2 on GIRK activity in CG neurons. We employed two forms of PIP2: one containing the dioctanoyl (DiC8) and the other containing mostly the arachidonic/stearic acid (AAst) side chains. PIP2(AAst) is purified from bovine brain and represents an endogenous form of PIP2. In inside-out patches, both forms of PIP2 (2-10 µM) failed to activate GIRK (n = 6) whether GTP was present or not (n = 7). When GIRK was activated with GTP (with baclofen in the pipette), 10 µM PIP2 applied to the bath solution produced an inhibitory effect, similar to that reported previously for atrial cells (Kim & Bang, 1999). However, when GIRK was activated first with GTPS, further application of PIP2 produced a marked increase in activity (Fig. 6A and B). Under our experimental conditions, 10 µM PIP2 produced a near-maximal effect on GIRK activity (K1/2, ~2 µM). In contrast to the effect produced by ATP, PIP2 produced an increase in the frequency of opening, without inducing long-lived openings, at all three concentrations of PIP2 tested (2, 5 and 10 µM). The lack of ATP-dependent gating of GIRK can be seen in the expanded current tracings for both forms of PIP2 (Fig. 6C and D). The duration histogram showed that GIRK openings could be fitted by a single exponential function either in the presence or absence of PIP2. The PIP2-induced changes in GIRK activity and kinetics were not significantly affected by application of the same cytosolic fraction that completely inhibited the ATP-induced changes in GIRK kinetics (Fig. 6E). The different effects of ATP and PIP2 on GIRK could also be demonstrated in the same patch. For example, in inside-out patches, ATP was applied after activating GIRK with GTPS. As before, ATP produced a 4.6 ± 0.6-fold increase in channel activity and was associated with induction of long-lived openings (
1 = 2.6 ± 0.3 ms). After completely washing off the effect of ATP with cytosol, 5 µM PIP2 was applied to the same patch. PIP2 also caused an increase in GIRK activity but only an increase in the frequency of opening was observed with no change in open lifetimes (0.4 ± 0.1 ms; n = 3). Together, these results suggest that the strong stimulatory effect of ATP is not mediated by production of PIP2 in the membrane.
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Figure 6. Increase in GIRK activity produced by phosphatidylinositol 4,5-bisphosphate (PIP2) and lack of inhibition by cytosol A, PIP2(DiC8) (PIP2 containing the dioctanoyl side chain; 5 µM) was applied to inside-out patches after activating GIRK with GTP | ||
We tested whether ATP was still capable of producing its stimulatory effect on GIRK in the presence of PIP2. If the ATP effect is mediated via PIP2 production, addition of ATP to patches treated with 10 µM PIP2 should yield no further increase in GIRK activity. Figure 7A shows GIRK recordings from an inside-out patch of a CG neuron. GTPS was applied to activate GIRK and then 10 µM PIP2(DiC8) was added to increase GIRK activity. Further addition of 2 mM ATP resulted in a strong additional activation of GIRK and this was reversed by addition of the cytosol. Since 10 µM PIP2 produced a nearly maximal activation of GIRK under our experimental conditions, the additional activation produced by ATP is highly unlikely to be due to greater availability of PIP2. On average, PIP2(DiC8) produced a 3.2-fold increase in GIRK activity and further addition of ATP resulted in an additional 2.2-fold increase in activity, producing a total of ~7-fold increase above the level observed with GTPS alone (Fig. 7A). Similar results were obtained when PIP2(AAst) was used, as shown in Fig. 6B. Due to high GIRK activity in these sets of experiments, it was not possible to determine the presence of ATP-dependent gating following addition of ATP for all patches. However, in two patches showing lower GIRK activity, longer-lived openings were observed (1 = 2.4 and 2.6 ms). The complete reversal of the ATP effect by the cytosol was also a strong indication that the ATP-dependent gating had occurred. After application of the cytosol, only short-lived openings were present with a mean open time of 0.5 ± 0.1 ms (n = 5). The result that cytosol, added at the end of the experiments, brought the GIRK activity close to the level observed with PIP2 showed again that the cytosol did not inhibit PIP2-induced increase in GIRK activity.
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Figure 7. Increase in GIRK activity by ATP in the presence of PIP2 A, GTP | ||
We also examined the effect of PIP2 after maximal stimulation of GIRK activity with ATP. We predicted that, if the mechanisms of modulation were separate, PIP2 would further increase GIRK activity after GIRK had been stimulated with ATP. In CG neurons, 2 mM ATP produced a maximal stimulation of GIRK activity, as higher concentrations of ATP (4 mM) did not increase the activity further. In inside-out patches of CG neurons and in the presence of GTPS, application of 2 mM ATP elicited an increase in GIRK activity and this was associated with long-lived open times, as judged by single-channel QuB analysis (Fig. 8). Surprisingly, addition of PIP2(DiC8) or PIP2(AAst) failed to produce a significant change in GIRK activity or the open lifetimes (Fig. 8C and D). The long-lived openings initially induced by ATP were still present and the open lifetimes were not significantly changed by PIP2 (2.4 ± 0.3 ms vs. 2.6 ± 0.3 ms, n = 5). Finally, application of cytosol decreased GIRK activity as expected, and also caused the disappearance of long-lived openings, consistent with the inhibitory effect of the cytosol on the ATP-dependent gating. The mean open time after application of the cytosol was 0.6 ± 0.2 ms (n = 3). Together with the data shown in Fig. 7, these results are consistent with the idea that the ATP-induced increase in GIRK activity occurs via two separate pathways: one involving generation of PIP2 and another that does not.
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Figure 8. PIP2 fails to affect ATP-modified GIRK channels A, in these inside-out patches, ATP was applied to allow ATP-dependent gating to occur and then PIP2(DiC8) was added. Expanded current tracings show channel openings at various times as indicated by lower case letters. B, same experiment as in A except that PIP2(AAst) was used. Expanded current tracings are also shown to illustrate the effects of ATP and PIP2. C, bar graph showing the marked stimulatory effect of ATP and the lack of effect of PIP2 on GIRK activity. Cytosol reduced GIRK activity and removed long-lived openings. Each bar is the mean ± S.D. of 4-5 determinations. D, bar graph shows the effect of ATP, PIP2 and cytosol on mean open times. Each bar is the mean ± S.D. of 3-4 determinations. * Significant difference from the GTP | ||
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DISCUSSION |
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The objective of this study was to characterize GIRK expressed in CG neurons at the single-channel level and to examine the modulation of GIRK function by various cellular factors that have been reported to affect atrial and cloned GIRK channels. The lack of studies on GIRK in CG neurons at the single-channel level has probably been due to the presence of relatively high densities of background K+ channels. This background K+ current is partly inhibited by acetylcholine via muscarinic receptors (Watkins & Mathie, 1996; Boyd et al. 2000; Han et al. 2002). Our recent studies show that there are four main types of background K+ channels in rat CG neurons. Three of them belong to the tandem-pore K+ channel family (TASK-1, TASK-3 and TREK-2) and one is a K+ channel sensitive to changes in extracellular pH near the physiological range (Han et al. 2002). Based on differences in single-channel conductances, GIRK could be clearly distinguished from the background K+ channels in CG neurons.
Single-channel properties of GIRK in CG neurons
CG neurons express mRNAs for GIRK1 and GIRK2, but very little or no mRNA for GIRK4 (Karschin et al. 1996; Wickman et al. 2000). The cerebellar granular cell layer has also been reported to express mRNA for GIRK3 in the rat brain (Karschin et al. 1996). Therefore, the native GIRK in CG neurons may be the functional correlate of GIRK2 homomer, GIRK1/2, GIRK1/3 or GIRK2/3, as all channel configurations are functional and activated by (Kofuji et al. 1995; Krapivinsky et al. 1995a; Lesage et al. 1995; Velimirovic et al. 1996; Jelacic et al. 1999, 2000). Our study suggests that the GIRK channels in CG neurons are not GIRK2 homomers, which show variable single-channel conductance (20-40 pS) and have a very short open time duration (0.1 ms; Krapivinsky, 1995) similar to that of GIRK4 homomers (Krapivinsky et al. 1995a; Velimirovic et al. 1996). In cultured CG neurons used in this study, the single-channel conductance (34 pS) and the mean open time duration (0.5 ms) are similar to those of GIRK1/2, GIRK1/3 and GIRK2/3 (Jelacic et al. 1999, 2000). Since mRNAs for all three GIRK subunits are present in the cerebellar granular layer, all three heteromers could be formed and could potentially function at different sites in the neuron.
Modulation of GIRK by ATP and PIP2 involves separate pathways in CG neurons
The presence of short- and long-lived opening of GIRK in mammalian sinoatrial node and atrial cells has been observed since 1983 (Sakmann et al. 1983; Soejima & Noma, 1984). Later studies have shown that the two open states of GIRK may arise due to the action of intracellular ATP (Kim, 1991). It is thought that the effect of ATP on GIRK and on other inwardly rectifying K+ channels is due to elevation of PIP2 concentration via lipid kinases in the plasma membrane. (Huang et al. 1998; Sui et al. 1998). The negatively charged PIP2 is thought to interact with certain positively charged amino acids within the cytoplasmic domains of GIRK, and thereby produce changes in GIRK gating properties (Huang et al. 1998; Zhang et al. 1999). The strength of interaction between PIP2 and GIRK also seems to be important in determining the activity of the channel when activated by (Zhang et al. 1999). This is consistent with recent studies showing that agonists that bind to receptors coupled to phospholipase C, which degrades PIP2, reduce GIRK current (Kobrinsky et al. 2000; Cho et al. 2001; Lei et al. 2001). Thus, the functional role of membrane PIP2 in supporting GIRK activity seems clear. However, our results provide evidence that the effect of ATP on GIRK activity does not appear to be simply mediated by formation of PIP2.
Our data obtained from CG neurons support the hypothesis that the modulation of GIRK by ATP is different from that produced by PIP2 alone. (1) ATP prolonged the open time duration of GIRK (ATP-dependent gating) whereas PIP2 increased only the frequency of opening in CG neurons. This frequency effect was observed with two different forms of PIP2. (2) The effect of ATP on both GIRK activity and open time duration could be abolished by the cytosolic fraction. The cytosol had no effect on the PIP2-induced change in GIRK activity. (3) Following a near-maximal stimulation of GIRK by PIP2, addition of ATP produced an additional 2.2-fold increase in GIRK activity. (4) During the course of our experiments, we noted that the effect of ATP was slowly reversible upon washout (> 5 min) whereas that of PIP2 was quickly reversible (< 15 s). Taken together, these differences provide a strong argument in support of a mechanism for ATP that is different from that produced by PIP2 alone.
One finding that does not seem to support the existence of completely separate mechanisms for ATP and PIP2 is that PIP2 fails to increase GIRK activity following maximal stimulation by ATP. However, the result can be explained if the effects of ATP on GIRK included the action of newly generated PIP2. In such a case, the application of PIP2 after ATP would not be expected to further augment GIRK activity. As ATP may elevate the concentration of PIP2 in the membrane, two separate mechanisms are thus likely to be involved in ATP-induced increase in GIRK activity. The mechanism that involves generation of PIP2 is likely to increase the frequency of opening whereas the other mechanism causes prolongation of the open time duration. In three patches, the mean closed times in the absence of ATP were 6.4 ± 1.6 and 1.8 ± 0.4 ms (C2 and C3 in Scheme 1), whereas they were 4.7 ± 1.1 and 0.8 ± 0.2 ms in the presence of ATP. Thus, the shortening of the closed time duration caused by ATP suggests that the probability of activation of GIRK by ATP may have been increased via production of PIP2 in those patches. In other patches (Fig. 4), however, cytosol completely reversed the effect of ATP, suggesting that PIP2 was not involved. We hypothesize that, in these patches, ATP does not cause a significant increase in PIP2 concentration as the PIP2 level is already optimal. Thus, we speculate that the effect of ATP on GIRK activity will depend on the initial level of PIP2 in the membrane, and this could explain the relatively large variation in the ATP-induced increase in GIRK activity.
In atrial cell membrane patches, PIP2 was able to produce a small increase in the mean open time of GIRK, although this effect of PIP2 was much smaller than that produced by ATP (Kim & Bang, 1999). The present finding that PIP2 had no effect on the open time duration of GIRK in CG neurons is interesting. Since GIRK in CG neurons may be made up of GIRK1/2, GIRK1/3 and/or GIRK2/3 that are all functional (Kofuji et al. 1995; Jelacic et al. 1999, 2000), it is possible that PIP2 modulates GIRK heteromers with different subunit compositions in a different way. Such a difference, if present, may help us to identify which heteromeric GIRK is present in each cell type. In any case, our results in CG neurons prompt us to seek a novel mechanism for the ATP-induced changes in GIRK function, and possibly the function of other ion channels and transporters. One potential mechanism may involve phosphorylation. However, none of the protein kinase inhibitors of protein kinase A, protein kinase C and tyrosine kinase were able to prevent the ATP effect in atrial cells (Kim, 1991; Hong et al. 1996). In our preliminary studies, wortmannin (1 µM), an inhibitor of PI3 kinase, also failed to block the effect of ATP in atrial cells (n = 4). We have not tested the effect of protein kinase inhibitors in CG neurons. The phosphorylated state of GIRK has been shown to be necessary for GIRK responsiveness to G proteins (Medina et al. 2000) and cAMP-dependent protein kinase seems to augment GIRK activity (Mullner et al. 2000). How ATP augments GIRK activity and alters the kinetics of gating remains to be determined. As discussed below, the mechanism of ATP-dependent gating involves interaction with potent inhibitory molecules in the cell. Understanding the mechanism of action of the inhibitor may give us a clue as to how ATP works.
Role of a cytosolic inhibitor and its identity
The presence of the brain cytosol abolished ATP-induced changes in GIRK kinetics in CG neurons, as it did in atrial cells. This shows that GIRK in CG neurons is also modulated by the two intracellular factors: ATP and a cytosolic inhibitor. We proposed earlier that the inhibitory component in the cytosol might be a group of free fatty acids (Kim & Pleumsamran, 2000). This was based on the findings that the protease-treated cytosol was still active and only the lipid fraction extracted from the cytosol could mimic the cytosolic effect. Among 40 lipophilic molecules tested, only low concentrations (0.5-2 µM) of unsaturated free fatty acids such as oleic, linoleic, linolenic and arachidonic acids were effective in abolishing the ATP-dependent gating (Kim & Pleumsamran, 2000). However, the role of free fatty acids in the modulation of GIRK remains sceptical and unproven, as convincing evidence supporting this hypothesis is lacking. Whatever the true identity of the inhibitor, its effect clearly outweighs the stimulatory action of ATP if both exist, as they would, in intact cells. This is in keeping with the observation that the 'ATP-dependent gating' is mostly absent in cell-attached patches when GIRK is activated by baclofen in CG neurons or by ACh in atrial cells.
To prove that free fatty acids are involved in modulating GIRK function, it was necessary to selectively remove them from the cytosolic fraction. Our method was to use recombinant brain FABP known to bind long chain free fatty acids. FABPs are thought to be important in the uptake and trafficking of fatty acids and in the regulation of their cellular concentration. Putative roles of FABPs include carrying fatty acids from plasma membrane (or the site of synthesis) to target organelles, modulation of the activity of enzymes involved in fatty acid metabolism, and supply of fatty acids for myelination of nerves (Veerkamp et al. 1991). The main ligand of FABP is long chain fatty acids with a molar ratio between 0.4 and 1.3 mol mol-1 of FABP (Veerkamp et al. 1991). FABP is also able to bind other ligands such as acyl-CoA but with a lower affinity. These unique properties of FABP allowed us to further test whether free fatty acids are involved in GIRK inhibition.
The cytosolic fraction that we have prepared presumably contains FABP and free fatty acids in equilibrium with FABP bound with free fatty acids. When the cytosolic fraction is incubated with recombinant FABP, the equilibrium would shift such that [free fatty acids] decreases and [bound FABP] increases. Therefore, the result that addition of FABP to the membrane patch removed the cytosolic effect on GIRK strongly suggests that the cytosol-induced inhibition of GIRK is caused by molecule(s) that bind FABP. These molecules are most likely to be free fatty acids and/or their CoAs, as they are the ligands for FABP. Since free fatty acids have higher affinity for FABP than acyl-CoAs, it seems likely that free fatty acids are the endogenous inhibitors of ATP-dependent gating of GIRK.
Physiological significance
GIRK is an important target of several neurotransmitters in neurons in many parts of the brain, and its activation provides an inhibitory input to the synaptic information flow. The degree of GIRK activity is therefore critical in neuronal function, as cell excitability is dependent on K+ permeability. Our results show that GIRK in CG neurons are positively modulated by ATP via two distinct pathways, and negatively modulated by cytosolic factors that appear to be free fatty acids. Under normal conditions, the cytosolic inhibitor prevents GIRK from entering the ATP-dependent gating, and thus keeps the channels in the short-lived open state. With the notion that PIP2 in the membrane is important to keep GIRK responsive to , our results show that PIP2 primarily regulates the frequency of GIRK opening without affecting the open lifetime in CG neurons. The cytosolic inhibitor has no influence on the PIP2 effect upon GIRK. Therefore, agonists that activate phospholipase C and cause degradation of PIP2 would produce a reduction in GIRK activity (Braun et al. 1992; Kobrinsky et al. 2000; Cho et al. 2001; Lei et al. 2001; Meyer et al. 2001) by reducing the frequency of opening and not the open time duration. Agonists that activate phospholipase C via muscarinic (M1 and M3) receptors have also been reported to inhibit GIRK activity via protein kinase C (PKC)-induced phosphorylation in oocytes and HEK 293 cells (Stevens et al. 1999; Hill & Peralta, 2001; Leaney et al. 2001). Therefore, both depletion of PIP2 and activation of PKC may be involved in the reduction of GIRK activity.
GIRK activity in cell-attached patches with baclofen in the pipette is very low, despite the ability of the channels to be more than 100-fold more active. This suggests that normally the [PIP2] in the membrane is far from that which can cause maximal activation of GIRK. Thus, the low [PIP2] and presence of free fatty acids are most likely to be the cause of the low GIRK activity associated with short-lived openings in healthy CG neurons. If a pathological event alters the level of free fatty acids inside the cell, it could produce changes in GIRK activity and influence synaptic transmission in the brain. Perhaps conditions such as stroke, ischaemia and seizure that might raise the levels of free fatty acids in the cell could potentially alter brain activity by inhibiting GIRK activity and therefore increasing cell excitability. GIRK activity that is under the influence of ATP and free fatty acids is a very interesting and an important phenomenon that could be related to metabolism-excitability coupling inside the cell. Our future studies are aimed at understanding the molecular mechanisms by which ATP and free fatty acids modulate GIRK function.
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Acknowledgements
This work was supported by a grant from the National Institutes of Health (D. Kim, NIH HL55363), and by the postdoctoral fellowship programme of Korea Science and Engineering Foundation (J. Han and D. Kang). We thank Stephen D. Kim for assistance with the electrophysiological data analysis and preparation of figures, and Dr Seung-Geun Hong for assistance with preparation of FABP.
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