Published online Jun 1 2004. doi:10.1085/jgp.200409029
The Rockefeller University Press, 0022-1295 $8.00
JGP, Volume 123, Number 6, 663-683
Regulation of KCNQ2/KCNQ3 Current by G Protein Cycling
The Kinetics of Receptor-mediated Signaling by Gq
Byung-Chang Suh1,
Lisa F. Horowitz1,
Wiebke Hirdes1,
Ken Mackie1,2, and
Bertil Hille1
1 Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA 98195
2 Department of Anesthesiology, University of Washington School of Medicine, Seattle, WA 98195
Address correspondence to Dr. Bertil Hille, Department of Physiology and Biophysics, University of Washington School of Medicine, G-424 Health Sciences Building, Box 357290, Seattle, WA 98195-7290. Fax: (206) 685-0619; email: hille{at}u.washington.edu
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ABSTRACT
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Receptor-mediated modulation of KCNQ channels regulates neuronal excitability. This study concerns the kinetics and mechanism of M1 muscarinic receptor–mediated regulation of the cloned neuronal M channel, KCNQ2/KCNQ3 (Kv7.2/Kv7.3). Receptors, channels, various mutated G-protein subunits, and an optical probe for phosphatidylinositol 4,5-bisphosphate (PIP2) were coexpressed by transfection in tsA-201 cells, and the cells were studied by whole-cell patch clamp and by confocal microscopy. Constitutively active forms of G
q and G11, but not G13, caused a loss of the plasma membrane PIP2 and a total tonic inhibition of the KCNQ current. There were no further changes upon addition of the muscarinic agonist oxotremorine-M (oxo-M). Expression of the regulator of G-protein signaling, RGS2, blocked PIP2 hydrolysis and current suppression by muscarinic stimulation, confirming that the Gq family of G-proteins is necessary. Dialysis with the competitive inhibitor GDPßS (1 mM) lengthened the time constant of inhibition sixfold, decreased the suppression of current, and decreased agonist sensitivity. Removal of intracellular Mg2+ slowed both the development and the recovery from muscarinic suppression. When combined with GDPßS, low intracellular Mg2+ nearly eliminated muscarinic inhibition. With nonhydrolyzable GTP analogs, current suppression developed spontaneously and muscarinic inhibition was enhanced. Such spontaneous suppression was antagonized by GDPßS or GTP or by expression of RGS2. These observations were successfully described by a kinetic model representing biochemical steps of the signaling cascade using published rate constants where available. The model supports the following sequence of events for this Gq-coupled signaling: A classical G-protein cycle, including competition for nucleotide-free G-protein by all nucleotide forms and an activation step requiring Mg2+, followed by G-protein–stimulated phospholipase C and hydrolysis of PIP2, and finally PIP2 dissociation from binding sites for inositol lipid on the channels so that KCNQ current was suppressed. Further experiments will be needed to refine some untested assumptions.
Key Words: M-current • M1 muscarinic receptor • phospholipase C • magnesium • PIP2
Wiebke Hirdes's present address is Institut für Angewandte Physiologie, Universitätsklinikum Hamburg-Eppendorf, Universität Hamburg, D-20246 Hamburg, Germany.
Lisa F. Horowitz's present address is Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N., Seattle, WA 98109.
Abbreviations used in this paper: GppNHp, guanylyl-imidodiphosphate; GTP
S, guanosine-5'-O-(3-thiotriphosphate); IP3, inositol 1,4,5-trisphosphate; oxo-M, oxotremorine-M; PIP2, phosphatidylinositol 4,5-bisphosphate.
1 The present model has a single intracellular compartment that we often refer to as "cytoplasm." It represents the cytoplasm and the nucleus lumped together. 
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INTRODUCTION
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This paper concerns the kinetics of steps in G-protein signaling to ion channels. The first biochemical experiments on the G-proteins Gs, Go, Gi, and transducin identified a cycle of conformational changes that activates and deactivates G-proteins (Schramm and Selinger, 1984
; Birnbaumer et al., 1985; Stryer, 1986; Gilman, 1987; Ross, 1995). These test-tube studies used purified protein components to measure rates of action of GTP and GDP analogs, AlF4–, and Mg2+ on nucleotide binding, tryptophan fluorescence, and proteolytic susceptibility of the G-protein -subunit. They identified steps of guanine nucleotide exchange and guanine nucleotide hydrolysis. Similar kinetic steps were subsequently recognized in electrophysiological work on Go-, Gi-, and transducin-dependent pathways of ion channel gating (Sather and Detwiler, 1987; Breitwieser and Szabo, 1988; Pfaffinger, 1988). Using both G-protein–activated inward rectifier K+ channels in heart and cyclic-nucleotide–gated channels in photoreceptors, such early work revealed the physiological kinetics of G-protein signaling in intact cells. In the case of photoreceptors, kinetic models could then be formulated to describe the kinetics of phototransduction (for review see Arshavsky et al., 2002).
Our goal is to carry out a similar biophysical analysis on a signaling pathway that uses G-proteins of the Gq family. Biochemical experiments with purified Gq and its relatives have identified -subunits of the Gq family as the activators of phospholipase C-ßs (PLC-ß), the enzymes that cleave the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3) (Sternweis and Smrcka, 1992; Singer et al., 1997). During activation in vitro, Gq steps through a cycle of nucleotide exchange and nucleotide hydrolysis like that of other G-proteins. The kinetics of these steps have been studied with purified components in lipid vesicles (Mukhopadhyay and Ross, 1999); however, less is known about the physiological kinetics in cells (see e.g., Pfaffinger, 1988; Lopez, 1992). Here we have monitored downstream actions of PLC in intact cells as measures of Gq activation while perturbing the system with GTP and GDP analogs, AlF4–, and Mg2+. As an indicator of PLC activity, we use principally the modulation of an ion channel, but we also use the translocation of a fluorescent reporter protein that is a probe for both a substrate and a product of PLC. With electrophysiology and confocal imaging, we have been able to follow the kinetics of Gq signaling, from which we have begun to formulate a preliminary kinetic model of its time course.
Our main PLC indicator is the voltage-dependent M-current, which can be suppressed by activating M1 muscarinic receptors or other receptors linked to Gq (Brown and Yu, 2000). A role for G-proteins in M-current modulation was recognized early by an irreversible inhibition of the current when compounds known to activate G-proteins, such as guanosine-5'-O-(3-thiotriphosphate) (GTPS), guanylyl-imidodiphosphate (GppNHp), or AlF4–, were applied intracellularly with or without muscarinic agonists (Pfaffinger, 1988; Brown et al., 1989). Evidence has accumulated that the G-protein involved belongs to the Gq/11 family (Pfaffinger, 1988; Brown et al., 1989; Caulfield et al., 1994; Jones et al., 1995; Haley et al., 1998; Shapiro et al., 2000). It is likely that different G-protein subtypes of the Gq/11 family participate in the transmitter modulation of M-current depending on the cell, receptor subtype, and species (Simmons and Mather, 1991; Haley et al., 2000).
The primary signal for suppression and recovery of M-current is the G-protein–mediated hydrolysis and depletion of PIP2 in the plasma membrane via activation of PLC, followed by resynthesis of PIP2. Muscarinic inhibition of the M-current is blocked by an inhibitor of PLC, recovery from inhibition requires cytosolic hydrolyzable ATP, and recovery is blocked by inhibitors of phosphatidylinositol (PI) 4-kinase (Suh and Hille, 2002). The M-current can be depressed by depleting PIP2 with antibodies or with polycations, and it can be reactivated by perfusion of PIP2 after suppression by rundown or exposure to agonists of Gq-coupled receptors (Zhang et al., 2003).
The other indicator of PLC activity that we use here is a fluorescent probe that binds to PIP2 in the membrane and to IP3 in the cytoplasm. This protein construct (PH-EGFP), a fusion protein of the PH domain of PLC-
1 with enhanced green fluorescent protein, can be expressed in cell lines and observed by confocal microscopy in living cells (Stauffer et al., 1998; Varnai and Balla, 1998). In resting cells, with PIP2 in the plasma membrane and no cytoplasmic IP3, the PH-EGFP protein binds to the inner leaflet of the plasma membrane, with little elsewhere in the cell. After PLC has been activated, the probe migrates into the cytoplasm both because of the loss of membrane PIP2 and because of the generation of cytoplasmic IP3 when PLC is active (Stauffer et al., 1998; Hirose et al., 1999; Xu et al., 2003; unpublished data).
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MATERIALS AND METHODS
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Cells for Electrophysiology
We express the M-current from its KCNQ2/KCNQ3 (Kv7.2/Kv7.3) channel subunits in a cell line together with M1 receptors. Plasmids encoding the channel subunits, KCNQ2 (EMBL/GenBank/DDBJ accession no. AF110020) and KCNQ3 (EMBL/GenBank/DDBJ accession no. AF091247), provided by David McKinnon (State University of New York, Stony Brook, NY), were subcloned into the pcDNA3 expression plasmid (Invitrogen). A plasmid containing mouse M1 muscarinic receptor was provided by Neil Nathanson (University of Washington, Seattle, WA). Plasmids with cloned human RGS2 and constitutively active forms of human Gq (Q209L), human G11 (Q209L), and human G13 (Q226L) were obtained from the Guthrie Research Institute. The plasmids encoding KCNQ2 and KCNQ3 subunits, the muscarinic M1 receptor, and sometimes the G-subunits were transiently cotransfected into human tsA-201 cells (tsA; derived from HEK293 cells) using lipofectamine 2000 (Life Technologies), together with cDNA-encoding green fluorescent protein (GFP) as a marker for successfully transfected cells (Shapiro et al., 2000). The 2-ml transfection medium usually contained 0.1 µg of GFP cDNA and 1 µg of each of the other cDNAs. The next day, the cells were plated onto poly-L-lysine–coated coverslip chips, and fluorescent cells were studied within 2 d in electrophysiological experiments.
Confocal Imaging
For the fluorescence measurements of PLC activation, we used a fluorescent indicator of PIP2 and IP3. The tsA cells were cotransfected with 0.25 µg cDNA for PHPLC-1-EGFP (PH-EGFP) (gift of Tobias Meyer, Stanford), 1 µg cDNA for M1 receptors, and 2 µg cDNA for G-subunits, if used, then transferred to poly-L-lysine–coated glass coverslips. 1 or 2 d after transfection, the coverslips were mounted in a perfusion chamber designed for a Leica TCS NT inverted confocal microscope. Live cells were imaged using a 63x water objective at 23°C. Control images were obtained for 1 min before drug application. The agonist oxo-M (10 µM) was applied by gravity feed. Images were processed with Metamorph (UIC) and Igor Pro (WaveMetrics) to obtain the time course of the average fluorescence intensity F in a cytoplasmic region normalized to the average intensity for 30 s before agonist application Fo (F/Fo). In one control experiment, confocal immunocytochemistry was used with fixed cells and anti-KCNQ2 and -KCNQ3 antibodies to ascertain trafficking of KCNQ channel subunits, as described (Roche et al., 2002). The cells were observed with a BioRad MRC 600 microscope.
Current Recording and Analysis
The whole-cell configuration of the patch-clamp technique was used to voltage-clamp and dialyze cells at 22–25°C. Electrodes pulled from glass micropipette tubes (VWR Scientific) had resistances of 1.3–2.5 M
. The whole-cell access resistance was 2–4 M, and series-resistance error was compensated >60%. Fast and slow capacitances were compensated before the applied test-pulse sequences. When measuring the rates of induction and recovery from muscarinic inhibition of the current, we applied test and control solutions rapidly to the 100-µl chamber (flow rate of 1.5 ml/min) in the vicinity of the recorded cell. Tests using junction-potential measurements on an open pipette showed that solutions changed with a mean exponential time constant of 2.4 s, and the change began with a 4–8 s delay after the command that switches the valve. Thus, there is an uncertainty of at least 4 s about the time of beginning (but not the duration) of the agonist exposure. In the figures, the bar representing solution change is usually drawn 8 s after the electronic command occurred. In separate experiments, we estimated the dialysis time for substances included in the whole-cell pipette by measuring the time course of fluorescence rise of cells after breakthrough with a pipette containing the fluorescent dye, indo-1. For indo-1 (M.W. = 645) the diffusion had an exponential time constant, 
120 s. As GDP and GTP analogues have similar molecular weights (460–540), we assumed that they also exchange into the cell with the same time constant as indo-1.
Currents were monitored by holding the cell at –20 mV and applying a 500-ms hyperpolarizing step to –60 mV every 4 s. For brevity, we will call the current from expressed KCNQ2 and KCNQ3 subunits, KCNQ current. The amplitude of the KCNQ current usually was defined as the outward current at the –20-mV holding potential sensitive to block by 30 µM of the channel blocker linopirdine. Time courses give the KCNQ current plotted every 4 s, except where noted. The agonist oxo-M was always applied at 10 µM unless noted. In most experiments with pipette solutions containing nucleotide analogs, we waited >300 s after breakthrough before applying oxo-M to allow time for the dialysis of the analogues into the cytoplasm.
Solutions and Materials
The external Ringer's solution used for current recording and confocal observations contained (in mM): 160 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 8 glucose, adjusted to pH 7.4 with NaOH. The standard pipette solution contained (in mM): 175 KCl, 5 MgCl2, 5 HEPES, 0.1 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA), 3 Na2ATP, and 0.1 Na3GTP, titrated to pH 7.4 with KOH (free [Mg2+] estimated as 2.1 mM, WinMaxc program v2.05, www.stanford.edu/~cpatton/maxc.html). Variations on this solution are noted in text. The "Mg2+-free" pipette solution had 1 mM EDTA and no added Mg2+, corresponding to an estimated free [Mg2+] of 14 nM, assuming a 10 µM Mg2+ contamination. Pipette solutions said to contain 100 µM AlF4– had 100 µM AlCl3 and 10 mM NaF. Reagents were obtained as follows: oxotremorine methiodide (oxo-M) (Research Biochemicals); BAPTA (Molecular Probes); Dulbecco's minimum essential medium, fetal bovine serum, lipofectamine 2000, and penicillin/streptomycin (Life Technologies); ATP, GTP, guanosine-5'-O-(2-thiodiphosphate) (GDPßS), GppNHp, GTPS, GDP, linopirdine, adenosine-5'-O-(2-thiodiphosphate) (ADPßS), AlCl3, NaF, and atropine (Sigma-Aldrich).
Data Analysis
Data acquisition and analysis used Pulse/Pulse Fit 8.11 software in combination with an EPC-9 patch-clamp amplifier (HEKA). Further data processing and statistical analysis were performed with Excel (Microsoft) and Igor Pro. Time constants were measured by exponential fits. All quantitative data are expressed as mean ± SEM and the number of observations is shown in parentheses in the histograms. Comparison between two groups was analyzed using Student's unpaired t test, and differences were considered significant at a level P < 0.05.
Kinetic Modeling
When the experiments were finished, we sought to represent the results in a self-consistent kinetic model. Like Xu et al. (2003), who simulated cellular breakdown of PIP2, we used the Virtual Cell environment of the National Resource for Cell Analysis and Modeling, University of Connecticut Health Center (http://www.nrcam.uchc.edu). In this JAVA-based simulation environment, components and their reactions are added through a graphical interface, initial conditions are stated, and the ordinary differential equations are generated and integrated automatically in time by a variable time-step, fifth-order, Runge-Kutta-Fehlberg routine. The working model with control values of rate constants and initial conditions is available at that web page for public use and modification.
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RESULTS
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Gq Couples to PIP2 Hydrolysis and KCNQ Current Inhibition in tsA Cells
In our expression system, the exogenously expressed M1 receptors should couple to endogenous G-proteins of the Gq family, which would activate endogenous PLC. We have shown in this system that M1 receptor–coupled cleavage of PIP2 suppresses the KCNQ K+ current of exogenously expressed KCNQ2/KCNQ3 channels and evokes intracellular Ca2+ release by an IP3-dependent pathway (Shapiro et al., 2000; Suh and Hille, 2002). We start by confirming that PLC is coupled to Gq in these cells.
TsA cells were transfected with the PH-EGFP probe and M1 receptors, with or without constitutively active, mutant forms of G-protein subunits. As expected, the PH-EGFP probe, which has affinity for membrane PIP2 and cytoplasmic IP3, was concentrated mainly at the cell surface in unstimulated control cells (circumferential dark regions in top left panel of Fig. 1 A). Bath application of oxo-M led to a rapid translocation (time constant, = 13 s) of the fluorescent probe from the membrane to the cytoplasm (Fig. 1, A and B). This translocation was slowly reversed after removal of oxo-M, recovering on average by 63% in 100 s (Fig. 1 B). However, when cells were cotransfected with a constitutively active Gq subunit (Gq*), most of the PH-EGFP probe was already found in the cytoplasm in resting cells, and additional incubation with oxo-M did not change the distribution of fluorescence (Fig. 1, A and B). Similarly, transfection with a constitutively active mutant of another Gq family protein, G11*, induced a cytoplasmic distribution of PH-EGFP in resting cells, and there was no further movement of the probe with oxo-M. As a control, transfection with constitutively active G13* did not displace PH-EGFP from the membrane or prevent the translocation seen with oxo-M (although the induced translocation was weaker). These data indicate that exogenous Gq* and G11*, but not G13*, can couple to PLC to activate potent PIP2 hydrolysis in tsA cells.
Signaling via G-proteins can be depressed by protein regulators of G-protein signaling, RGS proteins. They bind to the activated G subunit and reduce signaling (Hollinger and Hepler, 2002). One RGS protein, RGS2, is selective for the Gq family and interferes specifically with receptor signaling mediated by Gq (Heximer et al., 1997, 1999; Bernstein et al., 2004). We used it here to further document the involvement of Gq in muscarinic signaling in tsA cells. As shown in Fig. 1 C, expressing RGS2 had little effect on the resting localization of PH-EGFP, which was concentrated at the plasma membrane. However, the RGS2-expressing cells showed no translocation of PH-EGFP from the membrane during oxo-M treatment (Fig. 1, C and D). Thus, RGS2 abolishes muscarinic signaling to PLC.
Parallel experiments were done using the KCNQ current as an indicator of PLC activation. The cells were transfected with M1 receptors, KCNQ subunits, and GFP instead of PH-EGFP as a transfection marker. Fig. 2 A, top left, shows a typical time course of KCNQ current as oxo-M is perfused in the bath for 180 s and then removed. The current is nearly fully suppressed within 12 s (sample points are 4 s apart) in oxo-M and recovers over several hundred seconds after oxo-M is removed. In the control cells, current at –20 mV averaged 923 ± 328 pA (n = 10) and was almost completely inhibited by oxo-M stimulation (Fig. 2, A and B). It recovered slowly to 572 ± 256 pA after washout of the agonist. By contrast, in cells expressing constitutively active Gq*, KCNQ channel activity was essentially absent. The currents were similar to those seen in tsA cells not transfected with KCNQ2/KCNQ3 subunits. One explanation for the lack of KCNQ current in these cells might have been that expression of Gq* prevented the trafficking of KCNQ channels to the cell membrane. This possibility was ruled out by control immunocytochemical experiments using antibodies against KCNQ2 and KCNQ3 proteins. In control cells, immunoreactivity for both antibodies was distributed almost entirely at the cell surface and superpositions showed complete overlap of KCNQ2 and KCNQ3 (Fig. 2 C). In Gq-expressing cells the distribution of immunoreactivity was indistinguishable from that in control cells. KCNQ currents were also absent with expression of G11*, but not with G13*. However, with G13* expression, the action of oxo-M was somewhat slowed and reduced, both as measured by PH-EGFP translocation and by KCNQ current suppression. Likewise, in electrophysiological experiments, RGS2 severely attenuated agonist-induced suppression of KCNQ currents (Fig. 2, D and E). Inhibition by oxo-M was only 16 ± 6% (n = 8). Taken together, these results show that our confocal and electrophysiological indicators of PLC activity do reflect the activity of G-proteins of the Gq family and that Gq* is sufficiently active to abolish KCNQ current and PH-EGFP binding to the plasma membrane.
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FIGURE 2. Constitutively active Gq proteins and RGS2 alter KCNQ current. (A) Representative time courses of whole-cell current in cells transfected with M1 muscarinic receptor and KCNQ2/KCNQ3 channel subunits, without (None) or with constitutively active G subunits (G*). Oxo-M (10 µM) was bath applied for 3 min, and the current measured at –20 mV every 4 s. The inset shows selected current traces (at times a, b, c) with a dashed line at zero current. (B) Summary of KCNQ current regulation during expression of constitutively active G-proteins. The relative current at the selected time points was measured (symbols) from the indicated number of cells, and the average value is presented as a horizontal bar. (C) Confocal immunocytochemical images of cells stained with anti-KCNQ antibodies. Images are shown in inverted contrast with fluorescence being dark. The four panels show anti-KCNQ2 and -KCNQ3 in control cells and anti-KCNQ2 and -KCNQ3 in cells cotransfected with Gq*, respectively. (D) Representative time course of whole-cell current in RGS2-transfected cells, showing a reduced response to oxo-M. (E) Summary of KCNQ current changes in RGS2-transfected cells.
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GDPßS Slows and Reduces Suppression of KCNQ Current
The canonical G-protein activation cycle starts with receptor (R)-catalyzed dissociation of GDP from the G-protein, followed by binding and, eventually, hydrolysis of GTP that regenerates the GDP-bound G-protein (Schramm and Selinger, 1984; Stryer, 1986; Gilman, 1987). This continual recycling can be interrupted if the transient, nucleotide-free form of the G-protein (G) binds other nucleotides or nucleotide analogs, competitively altering the course of activation (simplified in Scheme I)
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SCHEME I
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Previous studies on sympathetic ganglion neurons showed that intracellular dialysis of GDPßS slows and decreases receptor-mediated suppression of M-current (Pfaffinger, 1988; Brown et al., 1989), presumably by sequestering Gq in the inactive Gq·GDPßS form. Fig. 3 A illustrates such changes in our system, comparing tsA cells dialyzed with 0.1 mM GTP (control), or 1 mM GDPßS alone, or a mixture of the two. With the standard 0.1 mM GTP in the pipette, steady bath application of oxo-M suppressed the KCNQ current almost completely, with a time constant of 7.6 ± 1.7 s (n = 20) (time constants summarized in Fig. 3 B). With 1 mM GDPßS and no GTP in the pipette, the fast component of inhibition was slowed approximately sixfold ( = 44 ± 6 s; n = 7; P < 0.001 compared with GTP alone) and reached only 81 ± 5% after 5 min of oxo-M. In mixtures of GTP and GDPßS, the slowing was graded with the fraction of GDPßS. On the other hand, addition of GDP, GppNHp, or ADPßS to the pipette without GDPßS did not reduce either the rate or completeness of oxo-M–induced channel inhibition (Fig. 3 B). GDP alone is not expected to support receptor-mediated activation of G-proteins, but we suppose that GDP was phosphorylated to GTP as it entered the cytoplasm in these experiments (see Suh and Hille, 2002).
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FIGURE 3. GDPßS decreases muscarinic inhibition of KCNQ current. (A) Muscarinic modulation of KCNQ current with different combinations of GTP and GDPßS in the pipette solution. Oxo-M was applied for 5 min and the current is given as mean ± SEM values relative to the preapplication level (n = 5–20). (B) Summary of the time constants of muscarinic inhibition () with different nucleotide analogues in the pipette solution. *, P < 0.01 and **, P < 0.001, compared with 0.1 mM GTP alone.
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Cytoplasmic GDPßS slows all aspects of muscarinic inhibition: onset, turn off, and recovery. In control cells perfused with 0.1 mM GTP, a 4-s exposure to oxo-M suffices to suppress the KCNQ current by 89 ± 5% (n = 4); the suppression reaches a peak within a few seconds after oxo-M is washed off; and the current recovers within a few hundred seconds (Fig. 4 A, top left, open circles). With GDPßS, the same oxo-M treatment gives only 5–15% inhibition (filled circles). Muscarinic inhibition increases in a graded manner with longer oxo-M exposures, and a 40-s exposure is needed for half-maximal inhibition. Unexpectedly, with the shorter oxo-M exposures, the development of current suppression continued long after oxo-M was removed (Fig. 4 A, top). Even for short exposures, the inhibition kept developing with a 40-s time constant after oxo-M is removed (Fig. 4 B, bottom). In GDPßS-treated cells, it appears that the G-protein–signaling pathway is activated only weakly, yet it takes a long time to shut down, and recovery is weak and slow. We argue later that the delayed action can arise because when GDPßS dissociates from the inactive G·GDPßS complex, the free G-protein eventually becomes activated by binding GTP. An alternative interpretation would be that oxo-M takes tens of seconds to dissociate from the receptor. This is unlikely since after 40 s of 10 µM oxo-M, the control trace recovers much faster than the GDPßS trace, crossing it. An additional experiment, using only 0.2 µM oxo-M to mimic the smaller net current suppression seen with GDPßS, also showed rapid recovery (Fig. 4 A).
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FIGURE 4. GDPßS slows and reduces muscarinic inhibition and recovery of KCNQ current. (A) KCNQ currents during applications of 10 µM oxo-M lasting 4, 40, 80, or 180 s. The control cells (open circles and triangles) have the standard 0.1 mM GTP pipette solution. In each panel, the test cells (filled circles) have 1 mM GDPßS without GTP in the pipette solution. Each trace is a different cell. One extra control trace in the 40-s panel (open triangles) shows the action of 40 s of 0.2 µM oxo-M. (B) Summaries of the maximum amplitude and time constant of muscarinic inhibition of current. Oxo-M was applied for 4, 20, 40, 80, 180, or 300 s to cells dialyzed with the GDPßS-containing pipette solution.
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Removing Intracellular Mg2+ Retards and Reduces Suppression of KCNQ Current
Intracellular Mg2+ is necessary for activation of G-proteins. After GDP dissociates from the inactive G-protein and GTP or a GTP analogue binds, Mg2+ promotes a nearly irreversible conformational change to the active state and thereby stabilizes the nucleotide binding to the G-protein (Higashijima et al., 1987a,b). The conformational change includes loss of the Gß-subunits. Expanding part of simplified Scheme I gives Scheme II
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SCHEME II
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In addition, Mg2+ is necessary for the subsequent hydrolysis of GTP by the G-protein (Higashijima et al., 1987a). Note that in Scheme II, the first three states of the G-protein refer to the Gß heterotrimer whereas the final active state contains only G.
To look for similar roles for Mg2+ in receptor-mediated channel modulation, we dialyzed cells with Mg2+-free or low-Mg2+ pipette solutions and monitored oxo-M–mediated suppression of KCNQ current. As we have seen with the standard pipette solution containing 5 mM total Mg2+ (2.1 mM free), oxo-M normally reduces the current with a 7.6-s time constant. However, eliminating the Mg2+ and including 1 mM EDTA in the pipette slowed muscarinic inhibition fivefold, = 38 ± 5 s (P < 0.001 compared with control, n = 13) (Fig. 5 A, left) and delayed its onset. We argue later that the delay and slowing reflect the slowness of the last step in Scheme II in low Mg2+ so that it now takes many tens of seconds on average to activate a G-protein. Fewer active G-protein complexes are formed per unit time and activation of PLC is much reduced and delayed. Despite the slowing of inhibition, the Mg2+-free pipette solution reduced the final extent of inhibition only a little (Fig. 5 B).
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FIGURE 5. G-protein–mediated inhibition of current requires intracellular Mg2+. (A) Inhibition of currents in cells dialyzed with the 5-mM added Mg2+ pipette solution (open circles) or with EDTA and no added Mg2+ (filled circles). The pipette solutions contained 0.1 mM GTP (left) or 1 mM GDPßS (right). (B) Summary of muscarinic inhibition of the current after dialysis with different combinations of guanine nucleotide analogues and different Mg2+ concentrations. (C) Inhibition of current by a 20-s oxo-M stimulation with Mg2+-containing (control) or Mg2+-free (EDTA) pipette solution. (D) Summary of time constants () for muscarinic inhibition with different Mg2+ and Ca2+ concentrations in the pipette. Oxo-M was applied for 20 s. The Mg2+ values given are the added amounts; the free Mg2+ for these solutions is given in the text.
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Although the molecular mechanisms of action are different, the effects of Mg2+-free pipette solutions on the rate and amount of current suppression resembled those of GDPßS. They each delay one of the sequential steps toward the formation of the final active form of Gq after receptor activation. Interestingly, when the Mg2+-free condition and GDPßS dialysis were combined, oxo-M no longer inhibited KCNQ current (Fig. 5 A, right, and B).
The pipette Mg2+ concentration also affected recovery of KCNQ current after short oxo-M exposure (Fig. 5 C). With the standard Mg2+-containing pipette solution, 20 s of oxo-M treatment suppressed current rapidly, and then the current recovered on average to a level of 70% (n = 7) of the preagonist value by 7 min after washout of the oxo-M (see also Suh and Hille, 2002). However, when the intracellular Mg2+ was absent there was no recovery after washout of oxo-M. In these experiments, as with GDPßS, the onset of inhibition was delayed, the time course of inhibition ( = 46 ± 8 s, n = 5) was slowed, and suppression of current continued to develop long after oxo-M was removed. We interpret the prolonged action and the lack of recovery to a combination of slowed synthesis of PIP2 (Porter et al., 1988) and a slowed GTPase rate (Higashijima et al., 1987b) that prolongs the active state of a small population of Gq.
In the experiments of Fig. 5, A–C, the free Mg2+ in the pipette was reduced to the low nanomolar range by adding EDTA. Intermediate reductions could be obtained without EDTA by adding less than the standard 5 mM Mg2+ to the solution. Here ATP is the principal Mg2+ buffer. The first four solutions in Fig. 5 D have 5, 2, 1, and 0.3 mM added Mg2+ and 2,100, 110, 31, and 7 µM free Mg2+. With 110 µM free Mg2+, the time course and magnitude of oxo-M inhibition were nearly normal, but recovery was already somewhat reduced, whereas, with 7 µM free Mg2+, the inhibition was appreciably slowed (Fig. 5 D), the magnitude was still nearly normal, but recovery was nearly absent.
GTP Analogues and AlF4– Can Activate Gq Spontaneously
G-proteins can be activated slowly without receptor stimulation by exposure to poorly hydrolyzable GTP analogues (GTPS, GppNHp) or to AlF4–. After spontaneous dissociation of GDP from the G-protein, the poorly hydrolyzable analogues can react with free G as in Schemes I and II to capture the G-protein in a stable active form. On the other hand, AlF4– reacts directly with and requires the GDP-bound form to occupy the site for the phosphate of GTP (Bigay et al., 1987; Sondek et al., 1994; Scheme III)
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SCHEME III
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We measured the apparent rates of these reactions by including 0.1 mM of the GTP analogues or of AlF4– in the pipette, without GTP (Fig. 6 A, left). In each case, the current gradually became completely suppressed over 100–800 s (note differing time scales) without any applied agonist. When fitted with single exponentials, spontaneous suppression developed with time constants = 63 ± 4 s (n = 6) for AlF4–, 98 ± 7 s (n = 6) for GTPS, and 346 ± 29 s (n = 5) for GppNHp. AlF4– is the fastest, presumably because it does not require GDP dissociation (Scheme III). GppNHp is the slowest, indicating that it reacts more slowly with nucleotide-free G-protein than GTPS does (or forms a less efficacious active form). Addition of equimolar GTP together with the GTP analogue to the pipette slows and attenuates the action of GTPS and GppNHp strongly (Fig. 6 A, right). The slowing is graded with the ratio of GTP analogue to GTP (Fig. 6 B), reflecting a competition for nucleotide-free G as expressed in Scheme I. The action of AlF4– is not slowed by GTP, confirming that it does not compete for free G with GTP as in Scheme III.
The recordings in Fig. 6 began 20 s after the moment of breakthrough to the whole-cell configuration. In evaluating them, we need to consider that, during the first minutes, the concentration of the reagents dialyzing from the pipette is gradually increasing in the cytoplasm ( = 120 s for GTPS or GppNHp) and any endogenous nucleotide is leaving. The slowness of this diffusional exchange might have delayed the initial reaction with GTPS or AlF4–, but it probably did not affect GppNHp, which is intrinsically slower to react. One check was to repeat the GTPS experiments with different pipette concentrations of GTPS (Fig. 6 C). Indeed, the rate of suppression of KCNQ current was slower for 0.05 mM GTPS and faster for 0.3 and 1 mM GTPS (Fig. 6 C).
In agreement with Schemes I and II, the spontaneous suppression of KCNQ current with GTPS is slowed by including either GDPßS-containing or Mg2+-free solutions in the pipette (Fig. 7, A and B). Also in agreement with predictions, the spontaneous action of AlF4– is not slowed by GDPßS, although it is slowed by Mg2+-free pipette solutions.
Nucleotide Analogues Can Diminish or Potentiate Muscarinic Action
Dissociation of GDP, the first step of Schemes I and II, is catalyzed by activated receptors. The subsequent capture of the nucleotide-free G-protein by GTP and Mg2+ then increases the fraction of active G-proteins and hence the physiological output of muscarinic signaling. By trapping G-proteins in an inactive form, GDPßS diminishes the receptor-evoked physiological output, and by forming an active G-protein that is long lasting, poorly hydrolyzable GTP analogues augment the physiological output. In these ways, nucleotide analogues should shift the effective dose–response curve for the oxo-M–induced suppression of KCNQ current. Since AlF4– reacts with the GDP form of G-proteins in an agonist-independent manner, its effect on the dose–response curve would be different.
To determine agonist dose–response curves, we measured the ability of various concentrations of oxo-M to reduce KCNQ current when some of the GTP in the pipette was replaced by other analogs. Consider, for example, the three traces for addition of 0.1 µM oxo-M in Fig. 8 A. With GTP in the pipette (top), 0.1 µM oxo-M gives almost 50% reduction of KCNQ current. As expected, GDPßS (middle) decreased the apparent potency of 0.1 µM oxo-M in comparison to GTP, whereas GTPS (bottom) enhanced it greatly. Fig. 8 B shows the resulting dose–response curves for current inhibition measured after 3 min in agonist. The EC50 values for channel inhibition differ by more than two orders of magnitude: 1.1 µM, 103 nM, and 5 nM for GDPßS/GTP, GTP, and GTPS/GTP in the pipette solution. In each case, the rate of current suppression fell as the oxo-M concentration was decreased (Fig. 8, C and D). As in Fig. 3, the initial inhibition rate was greatly slowed when GDPßS was present in the pipette, whereas it was not changed with GTPS. Additional experiments showed that the effective dose–response relation also could be shifted to the left by GppNHp in the pipette solution (to 76 nM with 0.05 mM GppNHp + 0.05 GTP), but not by AlF4– (102 nM with 0.01 mM AlF4– + 0.1 mM GTP). Overall, these results emphasize the mechanistic differences of the G-protein reaction with AlF4– as compared with the reactions with GTP, GTPS, or GppNHp. They also reemphasize that the EC50 of the dose–response curve for physiological outputs is not a direct measure of receptor occupancy for a G-protein–coupled receptor. The EC50 is the bottom line of a long cascade of events and can be altered over orders of magnitude by agents that may have little effect on receptor occupancy.
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FIGURE 8. Nucleotide analogues shift the dose–response relationship for muscarinic inhibition of current. (A) Typical time courses of muscarinic inhibition of KCNQ current by different oxo-M concentrations when the pipette contains GTP or GTP plus a nucleotide analogue as marked. (B) Dose–response relations for inhibition after 180 s in various concentrations of oxo-M (n = 3–10). (C) The time constant for oxo-M inhibition depends on the oxo-M concentration and the presence of nucleotide analogues (n = 3–10). (D) The time constant for oxo-M inhibition does not depend on the GTPS/GTP ratio, which was changed by including concentrations of 0/100, 50/50, and 80/20 µM, respectively (n = 5–6).
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RGS2 Blocks G-protein–coupled Inhibition of KCNQ Current
Expression of RGS2 had profound effects on current modulation. It prevented the spontaneous suppression of KCNQ current that normally develops in cells dialyzed with the G-protein activators GTPS or AlF4– (Fig. 9 A). In addition, RGS2 potently attenuated the receptor-mediated suppression of current in cells dialyzed with GTPS, even allowing some recovery (Fig. 9 B). These results indicate that G-proteins of the Gq family (the targets of RGS2) are required for the actions of GTPS and AlF4– here.
A Working Model for Gq Coupling to KCNQ Current Inhibition
The putative signaling pathway from M1 muscarinic receptors to KCNQ current modulation is complex, and the perturbations we have made could affect several intermediate steps. It therefore seemed worthwhile to test our understanding by an explicit kinetic model incorporating all of the postulated steps (Fig. 10). We wanted to see how far the full range of our observations could be explained by assembling simple concepts and kinetic measurements from the literature into a larger model. We consider this model a first approximation to be refined by future experiments. It has three conceptual components: First, the G-protein cycle of Gq and several side reactions with nucleotide analogues determine the number of active G-proteins. Then, phosphoinositide synthesis and breakdown determine PIP2 levels. Finally, the PIP2 level in the membrane governs the KCNQ current amplitude.
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FIGURE 10. Kinetic model used to simulate muscarinic modulation of KCNQ current. The model comprises a G-protein cycle (top), PI metabolism (below), and interaction of PIP2 with KCNQ channels (bottom). Most steps in the model have conventional first-order or second-order chemical kinetics with rate constants summarized in Table I. Further details are given in the APPENDIX. The rate constant k40 is defined for the GTPase reaction from right to left, and all other rate constants (e.g., k10, k20, k30) are for reactions going from left to right. The reverse reactions, if they are present (see direction of arrowheads) take a negative sign, e.g., k–10, k–20. The model indicates dissociation of Gß from G during the Mg2+ binding step (step 30) and reassociation of Gß in the GTPase step (step 40) in lumped reactions. However, here we have ignored Gß as a species and do not keep track of its concentration. Therefore, none of the steps is kinetically affected by a "concentration" of Gß.
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Before examining the model, we summarize what it can do. It was able to reproduce the following phenomena qualitatively: the time course of inhibition and recovery of KCNQ current during oxo-M exposure; spontaneous inhibition by GTP analogs; the competition among nucleotide analogues such that GDPßS slows and diminishes all modes of G-protein activation, except AlF4–, and GTP opposes spontaneous inhibition by GppNHp; the shift of the dose–response curve by GDPßS and GTPS; and the slowing of all modes of activation by Mg2+. Another valuable result was that formulating the model made apparent that the rates of many essential intermediate steps are not independently characterized in the literature. It allowed us to recognize the gaps in our knowledge.
Formulating the Model and Choice of Rate Constants
The full G-protein cycle is described in the top part of Fig. 10. The reactions are those in Schemes I, II, and III, and the rate constants are given in Table I. The literature provides biochemical measurements that suggest constraints on appropriate values for resting and stimulated cells. Nevertheless, many individual rate constants have not been studied, and in several cases our model constrains them very little. Frequently, the value chosen is tightly linked to values of other parameters, so that groups of parameters scale together. If one of them is fixed, then several others fall into place. Better values will await additional measurements. We did not use an automatic fitting program, so the parameter values are chosen by trial-and-error. We assumed that similar reactions should have similar rate constants.
The initial dissociation of GDP from G·GDP, step 10, is catalyzed by receptor (R) occupancy. This is represented as k10 x (OxoSat + 0.002), where OxoSat is a simple saturation function for oxo-M binding to M1 receptors (Koxo = 8 µM), and 0.002 represents a minute basal activity of this step in resting cells (possibly constitutive receptor activity). In the absence of agonist, this spontaneous dissociation of GDP should be fast enough to allow GTPS to turn off KCNQ current in 100 s. The kinetics of step 10 assumes instantaneous equilibration of agonist with M1 receptors, ignoring time dependence of agonist association and dissociation. Since dissociation of oxo-M from receptors probably takes <1 s, these events would be too fast to resolve with our solution exchange system. The quench-flow biochemical measurements of Mukhopadhyay and Ross (1999) give rates measured for the GTPase cycle of Gq with maximal M1 muscarinic stimulation and in the presence of PLC in a vesicle assay with purified proteins. In their work, the maximal steady-state rate of breakdown of GTP during full receptor activation (all around the cycle) is 0.4 moles GTP s–1 per mole Gq, and the individual rate constants equivalent to our k10, k20, and k40 are 0.8 s–1, 5.3 x 104 M–1 s–1, and 5.3 s–1 (all corrected to 23°C using a Q10 of 2.5). We chose 0.5 s–1, 4.5 x 105 M–1 s–1, and 1.8 s–1 for the same rate constants. In reconstituted systems, the GTPase activity of Gq is accelerated almost 2,000-fold by interaction with PLC, i.e., PLC has a potent GTPase acceleratory activity for Gq (Ross, 1995; Biddlecome et al., 1996; Mukhopadhyay and Ross, 1999; Cook et al., 2000). In our model, the GTPase activity of active Gq (step 40) is given a value closer to that for a complex with PLC than for the GTP-bound G-protein alone. Step 30, the reaction of GTP-bound G-protein with Mg2+ was made fast and not rate limiting at 2,100 µM free Mg2+ (k30 = 3 x 103 M–1 s–1 here and 1.7 x 104 M–1 s–1 in Higashijima et al., 1987b), but when Mg2+ is lowered to the low micromolar level, that step becomes rate limiting.
The lower part of Fig. 10 shows the second part of the model involving PIP2 synthesis and turnover. Steps 1 and 2 are PI-4-kinase and PIP-5-kinase. Both steps depend on Mg2+ and ATP concentrations, but unlike the model of Xu et al. (2003), we have not included any acceleration of these lipid kinase steps upon receptor activation. PIP2 is recycled back to PIP by PIP2 5-phosphatase, step 5. Step 3, the breakdown of PIP2 by the enzyme PLC, is activated by the active forms of Gq. It is represented by a rate expression that is simply proportional to the fraction of all active G-proteins (fGactive) plus a small basal PLC activity (k03 x [fGactive + 0.00075]). This description ignores potential diffusional steps for occupied receptors to find G-proteins and for active G-proteins to find unoccupied PLC molecules. Our kinetic assumptions would be literally correct if the relevant receptors, G-proteins, and PLC molecules existed and remained in 1:1:1 stoichiometric complexes throughout, a concept that has been suggested for Gq and PLC (Biddlecome et al., 1996).
Finally, the readout of these events comes from KCNQ channels. The model assumes that PIP2 binds reversibly and relatively slowly (seconds) to saturable sites on KCNQ channels, step 6. Each channel has fourfold symmetry and probably binds a minimum of four PIP2 molecules per channel. We chose a power law with an exponent of 1.8 for the activation of the channel by the bound PIP2 (see APPENDIX).
The level of PIP2 needed to keep KCNQ channels active should not be far below the resting level in the membrane, since the lag before channels begin to close during strong receptor activation is short. Thus, we chose a midpoint for KCNQ activation at 43% of the resting PIP2 level, which meant that KCNQ channels are 72% activated at the resting PIP2 level. The resting PIP2 level should be enough to make several micromolar IP3 if broken down all at once. We used a value of 5,000 µm–2 for PIP2 (McLaughlin et al., 2002; Xu et al., 2003), enough to make 5 µM IP3 in the cytoplasm and nucleus. With a 10 µM oxo-M stimulus, most of the PIP2 should be hydrolyzed by 15 s so that channels close. After agonist action, enough of the PIP2 should be restored in 300 s so that channels can reopen. When PIP2 synthesis is stopped (as with wortmannin), PIP2 levels should decay over 15 min (Willars et al., 1998) and KCNQ current should run down in that time (Suh and Hille, 2002). We chose nearly matching resting synthesis and breakdown of PIP2 at a rate that would turn over the entire PIP2 pool with an exponential time constant of 100 s. About half of this turnover goes via a futile cycle of PIP2 5-phosphatase back to PIP. Net replenishment of PIP2 all the way from PI would have a longer time constant (>200 s). If every G-protein could be simultaneously in an active state, the PLC rate would increase 1,330-fold over the basal level and the PIP2 pool would be hydrolyzed with a time constant of 210 ms. With the rate constants chosen and a 0.1 mM GTP solution in the pipette, only 21% of the G-proteins are simultaneously in the active G·GTP·Mg2+ state during a saturating agonist application, and only 13% during application of 10 µM oxo-M.
The Virtual Cell environment used in running the model (see MATERIALS AND METHODS) uses a specific set of self-consistent units. Time is in seconds, and distance is in micrometers. Concentrations are in micromolar for cytoplasmic1 and extracellular molecules and in molecules per µm2 for membrane molecules. Translation from cytoplasmic concentrations to membrane concentrations for mixed reactions is done automatically and requires specification of a surface (µm2) to volume (µm3) ratio. The value we used, 0.6 µm–1, is appropriate, e.g., for a round cell of 10 µm diameter or a flattened square box 20 x 20 x 5 µm (with negligible organelle volume) and means that releasing 1,000 molecules µm–2 from the membrane would yield 1.0 µM in the cytoplasm and nucleus.
Results of the Model
We now describe the output of the model, starting with the control responses to oxo-M application. Fig. 11 A shows the modeled activation of Gq by a 5-s instantaneous step of 10 µM oxo-M. At rest, 99.9% of the 200 G-protein molecules per µm2 are in the inactive heterotrimeric G·GDP form. After the agonist step, a pool of active G-proteins develops with a half time of 0.49 s (reaching 26 active molecules per µm2 in the steady-state). In this steady-state, 46 GTP molecules µm–2 are being broken down per second, so 23% of the G-proteins are cycled from G·GDP to G, to G·GTP, to G·GTP·Mg2+, and back to G·GDP each second and 0.23 moles GTP s–1 is broken down per mole of Gq.
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FIGURE 11. Early events in receptor activation calculated from the model with control conditions. (A) G-proteins. A 5-s instantaneous application of 10 µM oxo-M (dashed lines) rapidly converts a fraction of the resting G·GDP pool (upper solid line, right axis) to the other G-protein forms (free G, G·GTP, and G·GTP·Mg2+, which is labeled Gactive here) (Gx, solid line, left axis). (B) Downstream effects. Time course of decline of inositol lipids PIP and PIP2 (solid lines) and KCNQ current (solid line with symbols at 4-s intervals to simulate experimental recordings) during a 10-s application of oxo-M (dashed lines). All values are normalized. Oxo-M rises and falls with a 2.4-s exponential delay (see APPENDIX).
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Fig. 11 B shows the consequences for PI turnover and KCNQ current. In this panel and in the remaining model figures, the rise and fall of the agonist (dashed lines) are assumed to have exponential time constants of 2.4 s as in our experiments (see MATERIALS AND METHODS and APPENDIX), rather than being instantaneous. After agonist is applied, the PIP2 is rapidly depleted (half time = 2.7 s) because PLC is active. In addition, PIP falls a little because PIP regeneration from PIP2 by PIP2 5-phosphatase slows as PIP2 is depleted. With a resting PIP2 pool of 5,000 µm–2 and an invariant PI 4-kinase producing 18 PIP molecules µm–2 s–1, PIP and PIP2 recover only slowly when agonist is removed. The KCNQ current responds to PIP2 changes nonlinearly and with a delay governed by the kinetics of the reversible phospholipid binding to the activation sites on the channel (step 06). Thus, the minimum of KCNQ current is reached after the minimum of PIP2. The literature contains no kinetic measurements that constrain our choice of rate constants for PIP2 binding. They simply must be fast enough to allow KCNQ current inhibition in 8 s.
Fig. 12 shows slowing and blocking of oxo-M action when a GTP-free, GDPßS solution exchanges into the cytoplasm (compare the experiments in Fig. 4 A). In these and subsequent simulations with internal reagents, the simulation included time-varying intracellular concentrations. Nucleotides are assumed to exchange with a 120-s time constant (see MATERIALS AND METHODS and APPENDIX) starting 300 s before the application of oxo-M. In this simulation, intracellular GTP is falling with time as GDPßS is rising. The ability of oxo-M to inhibit KCNQ current declines for both reasons, and with even longer oxo-M application (see Fig. 13 A), KCNQ current is already recovering in the presence of agonist because GTP has been dialyzed away. The model predicts the strong slowing of inhibition by GDPßS, but it does not predict the near lack of recovery, for which we suggest a biochemical explanation in DISCUSSION.
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FIGURE 12. Model calculations of GDPßS antagonizing muscarinic suppression of KCNQ current. The control (solid line with symbols at 4-s intervals) is calculated KCNQ current (left axis) for a 4-s agonist application. All other solid lines are current with 1000 µM GDPßS in the pipette assuming that dialysis started at t = –210 s. Oxo-M (dashed lines and right axis) is applied for 4, 40, 80, or 180 s starting at t = 90 s. Conditions chosen to mimic Fig. 4 A.
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FIGURE 13. Model calculations of low Mg2+ antagonizing muscarinic suppression of KCNQ current. (A) Breakthrough is at t = –260 s and agonist is applied for 300 s starting at t = 40 s. The four pipette solutions are the standard 5-mM added Mg2+ solution (Control) or Mg2+-free pipette solution (EDTA) with 0.1 mM GTP, or with 1 mM GDPßS. Conditions chosen to mimic Fig. 5 A. (B) Prolongation of G-protein activation in low Mg2+. Agonist is applied for 20 s starting at t = 40 s with control or Mg2+-free (EDTA) pipette solutions. Conditions chosen to mimic Fig. 5 C.
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Fig. 13 simulates Mg2+-free intracellular solution (compare the experiments in Fig. 5, A and C). It is clear that the model predicts slowing and depression of agonist action (Fig. 13, A and B) and some continuing development of agonist action well after the agonist has been removed (Fig. 13 B). The low Mg2+ also intensifies the block by GDPßS (Fig. 13 A), but not as completely as was seen experimentally (Fig. 5 A).
Fig. 14 simulates the spontaneous inhibition of KCNQ currents with dialysis of intracellular GTP analogues or AlF4– (compare the experiments in Fig. 6). Unlike in the experiments, spontaneous inhibition has a sigmoid time course for all three reagents. The figure shows that if 0.1 mM GTP is included in the pipette solution, the action of GppNHp is greatly slowed. As in the experiments, GTP did not affect the action of AlF4– in the model (unpublished data).
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ABSTRACT
INTRODUCTION
