Pii: s0028-3908(02)00042-4

Mechanisms of GABA receptor blockade by millimolar concentrations of furosemide in isolated rat Purkinje cells Sergey N. Kolbaev ∗, Irina N. Sharonova, Vladimir S. Vorobjev, Vladimir G. Skrebitsky Brain Research Institute, Russian Academy of Medical Sciences, Moscow 103064, Russia Received 29 October 2001; received in revised form 27 February 2002; accepted 13 March 2002 Abstract
The action of diuretic furosemide on the GABAA receptor was studied in acutely isolated Purkinje cells using the whole-cell recording and fast application system. Furosemide blocked stationary component of GABA-activated currents in a concentration-dependent manner with IC value Ͼ 5 mM at Ϫ70 mV. The inhibition was rapid in the onset, fully reversible and did not require drug pre-perfusion. The termination of GABA and furosemide co-application was followed by transient increase in the inwardcurrent ‘tail’ current, which was not observed when furosemide was continuously present in the solution. The degree of furosemideblock did not depend on GABA concentration. Furosemide block increased with membrane depolarization. Five millimolar furosem-ide depressed GABA currents by 32.4Ϯ1.3% at –70 mV and by 76.7Ϯ5.0% at +70 mV. Analysis of the voltage dependence ofthe block suggests that furosemide binds at the site located within GABAA channel pore with a dissociation constant of 5.3Ϯ0.5mM at 0 mV and electric distance of 0.27. Our results provide evidence that furosemide interacts with Purkinje cell GABAAreceptors (most probably composed of α1β2/3γ2 subunits) through a low affinity site located in channel pore and suggest thatfurosemide acts as a sequential open channel blocker, which prevents the dissociation of agonist while the channel is blocked. 2002 Elsevier Science Ltd. All rights reserved.
Keywords: GABA; Cerebellum; Furosemide; Patch-clamp; Open channel block 1. Introduction
(Sieghart, 1995). The subunits have similar transmem- GABA receptors mediate the majority of fast inhibi- brane topology with an ~200 amino acid NH -terminal tory neurotransmission in the mammalian brain. Struc- extracellular domain, four closely spaced membrane- turally, GABA receptors are presumably heteropentam- spanning domains (TM1–TM4), a long and sequence- eric complexes and are assembled by combining variable intracellular loop between TM3 and TM4 and homologous subunits which enclose an integral anion a short extracellular COOH terminus. GABA receptors channel (Nayeem et al., 1994; Tretter et al., 1997). Seven are regulated by several classes of modulatory com- pounds one of the most receptor-subtype specific of identified in mammals (α, β, γ, δ, ε, π and θ) [for review, which is furosemide. Furosemide is a loop diuretic exert- see Kardos (1999); Sieghart et al. (1999)]. Several of the ing its action by interfering with the Na+/K+/2ClϪ co- subunit families have multiple subtypes (α1–6, β1–3, transport system in the lumen membrane (Greger and γ1–3). The majority of native receptors are formed by Wangemann, 1987). This diuretic compound, has been combination of αβγ or αβδ subunits (McKernan and Whiting, 1996). The particular subunits of which recep- eliciting approximately 100-fold greater sensitivity for tors are composed determine the pharmacological α6β2γ2 and for α4β2γ2 receptors than for α1β2γ2 recep-tors (Knoflach et al., 1996; Korpi et al., 1995; Waffordet al., 1996). The domain required for the action of furo- semide was described to residue amino-terminal to TM1 Corresponding author. Tel.: +7-95-917-84-52; fax: +7-95-917- of the α 6 subunit (Fisher et al., 1997). By making α1/α6 E-mail address: [email protected] (S.N. Kolbaev).
chimeras Thompson et al. (1999) identified a transmem- 0028-3908/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 2 8 - 3 9 0 8 ( 0 2 ) 0 0 0 4 2 - 4 S.N. Kolbaev et al. / Neuropharmacology 42 (2002) 913–921 brane region (209–279) responsible for the high furose- (Hamill et al., 1981). Glass recording patch pipettes were mide sensitivity of α6β3γ2 receptors. Furosemide prepared from filament-containing borosilicat tubes appears to reduce GABA receptor channel current in a using a two-stage puller. The electrodes, having resist- non-competitive manner (Inomata et al., 1988), but the ance of 2–3 MOhm, were filled with recording solution mechanisms by which furosemide suppresses GABA- of the following composition (in mM): 140 CsCl, 0.5 evoked current is unknown. Some studies indicate that CaCl , 4 MgCl , 10 HEPES, 5 EGTA, 4 ATP-Na (pH furosemide acts as ion channel blocker (Inomata et al., adjusted to 7.2 with CsOH). Recordings were carried out 1988). Other data support its allosteric regulation of the at room temperature (20–23°C) using an EPC 7 patch- GABA receptors (Korpi and Luddens, 1997). It is poss- clamp amplifier. Currents were filtered at 10 kHz, ible that mode of furosemide interaction with GABA sampled at 500 Hz, and stored on a computer disk.
receptor depends on subunit composition.
Cells were held at a membrane potential of Ϫ70 mV, The aim of the present study was to investigate the and I–V relationships were generated with test potentials from Ϫ70 mV to +70 mV by 20 mV intervals.
GABA receptors containing neither α6, nor α4 subunits and having low sensitivity to this drug. We have applied 2.3. Drug solutions and drug application patch-clamp technique to study the effect of furosemideon GABA receptors in freshly dissociated cerebellar A fast perfusion technique was used for application Purkinje cells. Our results indicate that furosemide inter- of agonist-containing solutions (Vorobjev et al., 1996).
acts directly with the GABA receptor pore and acts as Isolated Purkinje cells were first patch clamped and then a sequential open channel blocker, which prevents the lifted into the application system, where it was continu- dissociation of agonist when the channel is blocked.
ously perfused with control bath solution. The sub-stances were applied through two different glass capil-laries, ~0.2 mm in diameter. The delivery ports of the 2. Materials and methods
capillaries were positioned within 0.2 mm from the cell 2.1. Preparation of Purkinje cells under the study. For exposure the application system wasmoved so as to place the cell in the solution stream leav- All experiments were performed in compliance with ing one of the application capillaries. Exchange time was standards for use of laboratory animals and were measured at the open electrode tip by switching between approved by the USSR Academy Commission on Lab- solutions of different osmolarities. Rise time in these oratory Animals usage Control. Neurons were dis- experiments measured as the time elapsed from 10 to sociated acutely from cerebella of 2–3 week-old male 90% of the peak amplitude of the response was 20Ϯ3 Wistar rats as was described previously (Vorobjev et al., ms (n=6). The flow through each tube was gravity- 1996). Briefly, sagittal slices of cerebellum were incu- bated at room temperature for 1–6 h on a mesh near experiments GABA was applied for periods of 1 s, at the bottom of a beaker. The incubation solution had the 30–40 s intervals. Solutions were dissolved in extracellu- following composition (in mM): NaCl 125, KCl 5, CaCl lar buffer to the desired concentration from the following 1.5, MgCl 1.5, NaH PO 1.28, NaHCO 25, glucose 10, stock solutions: 100 mM GABA in H O and 200 mM Phenol Red 0.01%, it was continuously bubbled with furosemide in 200 mM NaOH. Furosemide was dis- carbogene (5% CO +95% O ). One at a time, slices were solved further in extracellular buffer and pH of final sol- transferred to the recording chamber and neurons were ution was controlled. All reagents were obtained from isolated by vertical vibration of a glass sphere, 0.7 mm in diameter, placed close to the surface of the slice(Vorobjev, 1991). Manipulation and cell identification was perfomed using an inverted microscope. IsolatedPurkinje cells were distinguished from other cerebellar Whole-cell records were analysed off-line using orig- cells based on their large cell bodies (ca. 20 µm) and inal homemade software or being exported as text files to characteristic pear shape attributable to the stump of the Prism (GraphPad Software, San Diego, CA) for further apical dendrite. The solution for dissociation and rec- analysis. Agonist concentration–response curve were fit ording had the following composition: NaCl 150, KCl 5, CaCl 2.7, MgCl 2.0, HEPES 10, pH adjusted to 7.4 where I is the peak current evoked by agonist concen- Voltage-clamp recording was obtained using the whole-cell configuration of the patch-clamp technique agonist concentration (100 µM GABA), EC is the con- S.N. Kolbaev et al. / Neuropharmacology 42 (2002) 913–921 centration giving half the maximal current, and n is the terized by single exponential time constants of 180Ϯ12 Hill coefficient. The curve fit was performed on an IBM ms and 252Ϯ24 ms for 2 and 5 mM furosemide, respect- PC-compatible computer using the program Prism ively (n=6). The relationship between the relative time (GraphPad Software). Data values are presented as We have made every effort to minimize animal dis- The time course of furosemide action is demonstrated comfort in our experimental procedures, and we have in Fig. 2C, where the degree of block was measured with used as few animals as necessary for this in vitro study.
intervals of 96 ms during co-application of GABA (2 µM) and furosemide (5 mM). One can see that thedegree of block is maximal at the beginning of co-appli- 3. Results
cation and reaches the equilibrium at the end of the 1-s application.
3.1. Effects of furosemide on GABA currents The termination of GABA and furosemide co-appli- cation was followed by transient increase in the inward Rapid application of GABA (2 µM) alone or together current (’tail’ current) (Fig. 1A). The onset of this cur- with furosemide induced in isolated Purkinje cells mem- rent coincided with the initiation of perfusion with nor- brane currents which were essentially completely mal extracellular solution. The amplitude and kinetics of blocked by bicuculline (20 µM, not shown), identifying the tail current were dependent on concentration of blocker and holding potential (Figs. 1 and 4). In the con- (Konnerth et al., 1990). Furosemide (1–5 mM) applied tinuous presence of furosemide the tail current was not together with GABA (2 µM), at Ϫ70 mV suppressed the responses to GABA, measured at steady state, in aconcentration-dependent manner. Representative super- 3.2. Blocking action of furosemide at different agonist positions of the currents induced by 2 µM GABA (control) or by GABA and furosemide co-applied at dif-ferent concentrations are shown in Fig. 1A. The greatest To determine whether inhibition by furosemide inhibition observed was 32.4Ϯ1.3% at 5 mM furosem- involved a competitive interaction with the channel acti- ide. Due to low solubility of furosemide full concen- vation by agonist, we examined its effect on the concen- tration-response curve could not be constructed. By tration-response relationship for GABA (Fig. 3).
itself, furosemide (up to 5 mM) had no action on resting The percent of block did not depend on the GABA concentration (Fig. 3B). Furosemide (5 mM) inhibited The effect of furosemide developed quickly, and was easily reversible. In order to test whether the blocking 27.8Ϯ3.2% (n=4), 36.5Ϯ1.5% (n=4) and 28.6Ϯ3.0% action of furosemide required activation of ion channel (n=4) at GABA concentrations of 2, 5, 10 and 50 µM, gating by agonist, furosemide was applied for 20–30 s respectively. Representative superpositions of the cur- prior to a concentration-jump application of GABA in rents elicited by GABA alone used at different concen- the continued presence of furosemide. Records on Fig.
trations (control) or by GABA co-applied with 5 mM 2 demonstrate that GABA currents evoked in the con- furosemide are illustrated in Fig. 3A. Furosemide does tinuous presence of 5 mM furosemide were indis- tinguishable from those in co-application experiment illustrated in Fig. 3C; the GABA concentration-response except for the kinetics of the recovery of responses. Pre- curve performed in the presence of 5 mM furosemide treatment of the neuron with furosemide (up to 5 mM) value of 2.9Ϯ0.1 µM with Hill coefficient did not affect the response to GABA when the agonist of 2Ϯ0.1 (n=4), which is close to the values determined was applied immediately after termination of washout of the antagonist, suggesting that furosemide effect does coefficient 1.8Ϯ0.1, n=4) (p values were 0.99 for EC50 Simultaneous application of furosemide with GABA (2 µM) caused a decrease in the rate of GABA receptor 3.3. Furosemide block at different holding potentials activation. Under control conditions, 2 µM GABAinduced a reproducible non-decreasing GABA response.
To determine the voltage dependence of furosemide The onset of this response was fitted by one exponential antagonism, block of the response to GABA was investi- with a time constant (t ) of 120Ϯ8 ms (n=6). Furosem- gated at several different membrane potentials. Fig. 4A ide (2–5 mM) slowed the activation time of GABA (2 illustrates the degree of block produced by 5 mM furose- µM) response. This effect was concentration-dependent.
mide on the current elicited by 2 µM GABA at Ϫ70, The time course of the rising phase of currents during Ϫ50, Ϫ30, Ϫ10, +10, +30, +50 and +70 mV. The co-application of GABA and blocker could be charac- GABA current–voltage relationship was roughly linear S.N. Kolbaev et al. / Neuropharmacology 42 (2002) 913–921 Furosemide blocks GABA-mediated currents in a dose-dependent manner. (A) Representative currents in acutely isolated cerebellar Purkinje cell induced by 2 µM GABA. The control current (lower trace) is superposed with the current induced by GABA co-application with differentconcentrations of furosemide (2, 3 and 5 mM). GABA applications are marked by the line and furosemide by the open bars above the currenttraces. The rise phase of response was adequately described by a single exponential function, and the smooth lines superimposed on the experimentalpoints represent an exponential function fit to the data. The time constants (ton ) are shown to the right of the curves. (B) Concentration–responseanalysis for block of GABA-activated currents by furosemide. Data points represent individual values from six cells normalized with respect tothe current obtained in control condition (2 µM GABA, Ϫ70 mV); solid line, the average data fitted by the equation: I 1 / (1 ϩ ([FURO] / IC50)nH), were Iblocked is the current induced by co-application of GABA (2 µM) and furosemide at a given concentration and Inon-blocked is the amplitude of steady-state current evoked by GABA (2 µM), IC50 is the concentration of the furosemide producing a half-maximalblock of GABA-mediated responses, and nH is the Hill coefficient. Fitting the data with this logistic equation, where maximal percent of blockwas set at 100% produced an apparent IC50 of 8.9Ϯ0.7 mM and Hill slope of 1.5Ϯ0.1. Current amplitude was measured at the end of application atthe moment marked by a dashed line. (C) The relationship between the relative activation time (ton-blocked/ton-control) and concentration of furosemide.
in the absence of furosemide, but showed significant 3.4. Woodhull analysis of furosemide block inward rectification in the presence of furosemide (Fig.
4B). Thus, the block of GABA-induced currents was The voltage dependence of block was further analysed greatest at the most positive holding potentials. At 5 according to the method of Woodhull (1973) which pro- mM, furosemide depressed stationary GABA current by vides a means of calculating the fraction of the trans- 32.4Ϯ1.3% at Ϫ70 mV and by 76.7Ϯ5.0% at +70 mV membrane field sensed by charged blocking ligand at its acceptor site. Furosemide contains a carboxyl group At all membrane potentials the co-application of which has a negative charge. Therefore, the molecule GABA and furosemide was associated with an appear- would be expected to carry a single negative charge at ance of the tail current following cessation of the appli- physiological pH. According to Woodhull’s model, two cation. The maximal relative amplitude of the tail current sets of GABA currents (steady-state currents in the measured from the level of the steady-state current was absence and presence of furosemide) were measured in observed when the degree of block was maximal — at the same cell at different membrane potential. Then the furosemide concentration of 5 mM and holding potential relation of the B value (B ϭ I brane potential (V) were approximated by: For estimation of voltage-dependence of tail current the absolute amplitude (from baseline) was measured 1 ϩ ([Furosemide] / K (0))∗exp(zdFV / RT) and normalized to GABA response (Fig. 4B). This nor- malized current-voltage relationship was similar to that where K (0) is the dissociation constant of the furosem- obtained for GABA alone. Reversal potential of tail cur- ide-binding site on the GABA complex at a transmem- rent was close to zero. Its absolute amplitude was brane potential of 0 mV, d is the measureless factor smaller than the amplitude of control steady-state current which reflects the fraction of the total electric field sensed at the binding site, z is the charge of furosemide S.N. Kolbaev et al. / Neuropharmacology 42 (2002) 913–921 The development of furosemied effects does not require pre-equilibration. The continuous presence of furosemide prevents the appearance of the tail current. (A) Traces show the effect of 5 mM furosemide on currents produced by 2 µM GABA. When furosemide remained after GABAapplication was stopped (trace 3) there was no tail current. (B) Superposition of the second and third traces presented on A. (C) The time courseof development of furosemide block. The relative degree of block (percentage of block) during co-application with GABA is marked by a solidline, percentage of furosemide block in the continuous presence of blocker is marked by dashed line. Mean values from four cells.
(=Ϫ1), F is the Faraday constant, R is the universal gas receptors. It displays a high affinity for inhi- constant and T is the ambient absolute temperature bition of currents elicited by GABA in recombinant [F / (RT) ϭ 0.03972 mVϪ1]. [Furosemide]Ϫfurosemide receptors containing α4 or α6 subunit and very low concentration (5×10Ϫ3 M). The values of K (0) and d affinity in α1β2γ2 receptors (the order of magnitudes of were determined as a result of nonlinear regression. The is ~100 µM for α4- or α6-contanig receptors versus results of fitting and experimental data are shown on Fig.
~10 mM for α1, α2-, α3- and α5-contaning receptors) 4C. The best fitted values of K (0) and d were 5.3Ϯ0.3 (Knoflach et al., 1996; Korpi et al., 1995; Wafford et mM and 0.27Ϯ0.03, respectively. The K (Ϫ70 mV) al., 1996). In situ hybridization studies have show strong was evaluated using equation (Woodhull, 1973): expression of the α1, β2, β3 and γ2 mRNA in rat cer-ebellar Purkinje cells (Laurie et al., 1992). The func- K (V) ϭ K (0)∗expͩzdFVͪ.
tional properties of GABA receptors of Purkinje cells also suggest the presence of α1, β2, β3 and γ2 subunits.
The derived value (10 mM) is in agreement with the These include the high sensitivity to zolpidem (Itier et value obtained from the concentration-block experiment al., 1996; our unpublished observation), indicating the shown in Fig. 1, which was obtained at a holding poten- presence of α1 and γ2 subunits, the high potency of lore- tial of –70 mV (Ͼ 5 mM). The results of such analysis clezole (our unpublished data), suggesting the presence provide evidence for binding of furosemide to a site of β2/3 subunits, and the relatively low sensitivity to within the membrane electrical field.
Zn2+ (Sharonova et al., 2000), reflecting the presence of γ subunits. All these facts imply that the majority offunctional GABA receptors in cerebellar Purkinje cells 4. Discussion
most probably have α1β2/3γ2 subunit composition.
As expected, these receptors display a very low affin- 4.1. Blocking action of furosemide ity for furosemide. The direct measurement of the IC50value for furosemide inhibition was hampered by its In the present study we have characterized the effects solubility limit. However, estimation of an IC value Ͼ5 mM, which is in agreement with IC cerebellar Purkinje cells. Furosemide was reported to be measured by other authors on recombinant α1β2/3γ2 receptors expressed in different heterologous systems S.N. Kolbaev et al. / Neuropharmacology 42 (2002) 913–921 The degree of furosemide-induced block does not depend on GABA concentration. (A) Responses to 2, 10 and 50 µM GABA co-applied with 5 mM furosemide (smaller responses) and corresponding control responses. All responses are from a single cell. GABA application is markedby a line and furosemide exposure by an open bar above the traces. Current amplitude was measured at the end of application at the momentmarked by a dashed line. (B) Percent of block by furosemide (5 mM) does not dependent on GABA concentration. (C) Furosemide does notmodify the EC value of GABA. Concentration-response curve to GABA obtained in control (squares) or presence of 5 mM furosemide (triangles).
Data points represent averages from four cells. Data are normalized with respect to the response to 50 µM GABA in the absence of furosemide.
The data are fit with eq. (1): control: EC =2.8Ϯ0.1 µM, n =1.8Ϯ0.1 and at 5 mM furosemide: EC =2.9Ϯ0.1 µM, n =2Ϯ0.1.
(Fisher et al., 1997; Korpi et al., 1995). Thus, furosemide antagonist (Inomata et al., 1988; Kumamoto and Murata, interacts with low-affinity site/sites on GABA receptors 1997). The non-competitive nature of furosemide antag- onism is proved by concentration–response data. We We have demonstrated that furosemide induces con- have not observed the decrease in the percentage of furo- centration and voltage-dependent suppression of GABA- semide antagonism when the concentration of GABA activated Cl–currents. Under experimental conditions used (co-application with GABA during 1 s) furosemide modified by the presence of furosemide also implying a effects were mediated by its direct interaction with non-competitive mechanism of blockade.
GABA receptor. This statement is supported by the fol- Analysis of blocking action of furosemide on GABA receptors in Purkinje cells has led us to two main con-clusions: 1. no current was evoked by isolated 1–30 s application 1. furosemide is an open channel blocker of these 2. co-application of GABA and furosemide in the pres- ence of bicuculline did not elicit any response; and 2. channel block by furosemide prevents dissociation of 3. furosemide did not change the reversal potential of 4.2. Furosemide is an open-channel blocker Therefore, the effect of furosemide in the present experiments is neither mediated by the changes of mem- The mechanism of furosemide-induced suppression of brane properties due to non-specific binding nor by the GABA-gated chloride conductance is largely unknown.
shift in reversal potential, as would result from an altered We provide evidence that furosemide inhibits GABA currents in cerebellar Purkinje cells by a mechanism of The results of the present study support the suggestion open channel block. The following observations support that furosemide is a non-competitive GABA S.N. Kolbaev et al. / Neuropharmacology 42 (2002) 913–921 brane region of the channel. The present study has dem-onstrated that mediated whole-cell currents by furosemide was voltagedependent. It means that the weak block occurring atnegative potentials is significantly increased when thecell is brought to depolarized potentials. Analysis of thesteady-state fractional block values for furosemideaccording to the method of Woodhull provided an esti-mated electrical depth of the binding site of 0.27. Thisparameter represents the fraction of the membrane elec-trical field that is sensed by furosemide at its bindingsite. The value of 0.27 suggests that the putative channelbinding site for these molecules must be located in thefirst third of the field across the ionic pore (assumingthat electrical field across the lipid bilayer is constant).
Thus, our results suggest that furosemide antagonizes responses to GABA in Purkinje cells by entering andblocking the ion channel activated by the GABA recep- tors. This GABA antagonism by high furosemide con- centrations differs from that of α6-containig receptorsince the interaction of furosemide with the high affinitybinding site is voltage-independent (Korpi et al., 1995), Block of GABA induced currents by furosemide is voltage- suggesting an allosteric mechanism of inhibition. The dependent. (A) Responses to 2 µM GABA at holding potentials Ϫ70, Ϫ50, Ϫ30, Ϫ10, 10, 30, 50 and 70 mV in control (left) and during low affinity site is unlikely to coincide with the picro- co-application with 5 mM furosemide (right). Holding potential was toxin-sensitive binding site since the latter is located adjusted 10 s before GABA exposures during time indicated by lines much deeper into the channel pore (Xu et al., 1995; Zho- above the traces. Offset current in the traces is subtracted. (B) The current-voltage relationships for the control (2 µM GABA) currents(circles), the currents blocked by 5 mM furosemide (squares) and forthe tail current (triangles). The IϪV curves constructed from measure- 4.3. Furosemide prevents the GABA channel from ments in four cells. Responses obtained at different voltages were nor- malized to the response at Ϫ70 mV in the absence of furosemide. (C)Woodhull analysis of furosemide block. Normalized block is plotted Two main mechanisms of the open channel block versus for the holding potential. The data are fitted using eq. (2) after have been described for ligand-gated ion channels: ‘trap- normalizing. The fraction of the membrane field sensed by furosemideis 0.27Ϯ0.03 with IC ping’ and sequential block. Trapping channel blocker permits agonist dissociation and channel closure whilethe blocker is bound in the activated channel. It results 1. no inhibitory action of furosemide on closed channel in trapping of the blocker in the channel (Blanpied et al., 1997; MacDonald et al., 1987, 1991; Samoilova et 2. furosemide block of GABA-induced current was volt- al., 1999). When the blocker is trapped in the channel pore, the recovery occurs only in the presence of agonist.
In the sequential scheme, the channel cannot close while Our data show that furosemide binds rapidly to the blocked (Adams, 1976; Antonov and Johnson, 1996; open, but not to the closed (unliganded) state of GABA Benveniste and Mayer, 1995; Neher and Steinbach, receptor. Pre-incubation in furosemide prior to co-appli- 1978; Neher, 1983; Sobolevsky et al., 1999).
cation of agonist and furosemide did not influence either The kinetic scheme for sequential channel-blocking the onset kinetics of GABA response or the block ampli- model implies that agonist (A) cannot dissociate from tude, suggesting essentially no blocking action of furose- its binding site on a receptor (R) when the ion channel mide application on closed channel. Thus, the results of our experiments indicate that block of GABA channels by furosemide is use-dependent. This is typical for the action of drugs that need to access the channel pore in This scheme also implies that prior to dissociation of order to exert their blocking action. Open channel block- the agonist, channels which are blocked must return to ers generally are known to exhibit a marked voltage- the closed state via the open state. In this case one may dependent inhibition. Voltage dependency suggests that expect the appearance a tail currents developing upon the drug binds to a site sensitive to electric field across removal of agonist and blocker. Tail currents of that type the membrane bilayer, i.e. a site within the transmem- were observed after termination of the co-application of S.N. Kolbaev et al. / Neuropharmacology 42 (2002) 913–921 NMDA or aspartate together with a number of NMDA anism of ligand-gated ion channels, including GABAA Sharonova, 1994), 9-aminoacridine (Benveniste and In conclusion, the results of our experiments strongly Mayer, 1995; Costa and Albuquerque, 1994), tetrabutyl- suggest that the voltage-dependent block produced by ammonium (Koshelev and Khodorov, 1995).
furosemide occurs by an open channel blocking mech- Our results provide evidence that similar mechanisms anism and conclusively demonstrate that it is a sequen- may underlie furosemide action on GABA currents: tial blocker that prevents the closure of the channel.
Therefore it is reasonable to use furosemide and itsderivatives to study the relationship between agonist block was fast and did not require the presence of anagonist; and 2. furosemide induced the appearance of a tail current after termination of its co-application with the agon- Acknowledgements
This work was supported by the Russian Foundation for Basic Research (Grant Nos 99-04-48429 and 00-04- The amplitude of the tail current was dependent on 04001) and Deutsche Forschungsgemeinschaft (Grant membrane potential and blocker concentration. The tail current was not observed when furosemide perfusionwas continued after GABA application was stopped. Theabove mentioned properties of furosemide-induced inhi- References
bition are consistent with the sequential model of openchannel block.
Adams, P.R., 1976. Drug blockade of open end-plate channels. The We have observed that furosemide slowed the onset Journal of Physiology (London) 260, 531–551.
of GABA-induced current in a concentration-dependent Antonov, S.M., Johnson, J.W., 1996. Voltage-dependent interaction of manner. This phenomenon also could be explained in open-channel blocking molecules with gating of NMDA receptors the framework of the sequential model. This model pre- in rat cortical neurons. The Journal of Physiology (London) 493, dicts that total open time/burst should be unchanged in Benveniste, M., Mayer, M.L., 1995. Trapping of glutamate and glycine the presence of a blocking drug (Neher and Steinbach, during open channel block of rat hippocampal neuron NMDA 1978). After dissociation of a drug, the channel passes receptors by 9-aminoacridine. The Journal of Physiology (London) back through the open state before closing. We can sup- pose that at the beginning of application furosemide Blanpied, T.A., Boeckman, F.A., Aizenman, E., Johnson, J.W., 1997.
induces the fast blockade of high proportion of open Trapping channel block of NMDA-activated responses by amantad-ine and memantine. Journal of Neurophysiology 77, 309–323.
GABA channels. Subsequent partial rescue from the Costa, A.C., Albuquerque, E.X., 1994. Dynamics of the actions of block may result from the prolongation of bursts and tetrahydro-9-aminoacridine and 9-aminoacridine on glutamatergic reopening of channels after dissociation of the blocker.
currents: concentration-jump studies in cultured rat hippocampal Another possible explanation for the described effect is neurons. The Journal of Pharmacology and Experimental Thera- that the blocker prohibits not only the channel closure, Fisher, J.L., Zhang, J., Macdonald, R.L., 1997. The role of α1 and but also does not allow the receptor to desensitize. A α6 subtype amino-terminal domains in allosteric regulation of γ- similar explanation has been proposed to explain the aminobutyric acid receptors. Molecular Pharmacology 52, 714– identical effect of tetrapentylammonium on NMDA channels (Sobolevsky et al., 1999). Further kinetic study Greger, R., Wangemann, P., 1987. Loop diuretics. Renal Physiology of this effect and simulation experiments are needed to Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981.
Improved patch-clamp techniques for high-resolution current rec- Kinetic studies on nicotinic (Neher, 1983) and NMDA ording from cells and cell-free membrane patches. Pflu¨gers receptors (Benveniste and Mayer, 1995) suggest that sequential channel block prevents escape of agonist from Inomata, N., Ishihara, T., Akaike, N., 1988. Effects of diuretics on its binding site. Similar mechanism seems to occur in GABA-gated chloride current in frog isolated sensory neurones.
British Journal of Pharmacology 93, 679–683.
Itier, V., Depoortere, H., Scatton, B., Avenet, P., 1996. Zolpidem func- The molecular mechanisms underlying agonist trapping tionally discriminates subtypes of native GABA during open channel block are not yet understood, but acutely dissociated rat striatal and cerebellar neurons. Neuropharm- this effect suggests a close coupling between confor- Kardos, J., 1999. Recent advances in GABA research. Neurochemistry opening/block and agonist dissociation. Experiments Knoflach, F., Benke, D., Wang, Y., Scheurer, L., Luddens, H., Hamil- with the channel blockers that prevent channel closure ton, B.J., Carter, D.B., Mohler, H., Benson, J.A., 1996. Pharmaco- could be useful for giving insight into the gating mech- logical modulation of the diazepam-insensitive recombinant γ-ami- S.N. Kolbaev et al. / Neuropharmacology 42 (2002) 913–921 methyl-4-isoxazolepropionate receptors in rat brain neurons. Neu- Konnerth, A., Llano, I., Armstrong, C.M., 1990. Synaptic currents in Sharonova, I.N., Vorobjev, V.S., Haas, H.L., 2000. Interaction between cerebellar Purkinje cells. Proceedings of the National Academy of copper and zinc at GABA receptors in acutely isolated cerebellar Purkinje cells of the rat. British Journal of Pharmacology 130, Korpi, E.R., Luddens, H., 1997. Furosemide interactions with brain GABAA receptors. British Journal of Pharmacology 120, 741–748.
Sieghart, W., 1995. Structure and pharmacology of γ-aminobutyric Korpi, E.R., Kuner, T., Seeburg, P.H., Luddens, H., 1995. Selective acidA receptor subtypes. Pharmacological Reviews 47, 181–234.
antagonist for the cerebellar granule cell-specific γ-aminobutyric Sieghart, W., Fuchs, K., Tretter, V., Ebert, V., Jechlinger, M., Hoger, acid type A receptor. Molecular Pharmacology 47, 283–289.
H., Adamiker, D., 1999. Structure and subunit composition of Koshelev, S.G., Khodorov, B.I., 1995. Blockade of open channels by GABAA receptors. Neurochemistry International 34, 379–385.
tetrabutylammonium, 9-aminoacridine and tacrine prevents chan- Sobolevsky, A.I., Koshelev, S.G., Khodorov, B.I., 1999. Probing of nels closing and desensitization. Membrane Cell Biology 9, 93– NMDA channels with fast blockers. The Journal of Neuroscience Kumamoto, E., Murata, Y., 1997. Action of furosemide on GABA- Thompson, S.A., Arden, S.A., Marshall, G., Wingrove, P.B., Whiting, and glycine currents in rat septal cholinergic neurons in culture.
P.J., Wafford, K.A., 1999. Residues in transmembrane domains I and II determine γ-aminobutyric acid type A receptor subtype- Laurie, D.J., Seeburg, P.H., Wisden, W., 1992. The distribution of 3 selective antagonism by furosemide. Molecular Pharmacology 55, receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. The Journal of Neuroscience 12, 1063–1076.
Tretter, V., Ehya, N., Fuchs, K., Sieghart, W., 1997. Stoichiometry MacDonald, J.F., Miljkovic, Z., Pennefather, P., 1987. Use-dependent and assembly of a recombinant GABA receptor subtype. Journal block of excitatory amino acid currents in cultured neurons by keta- mine. Journal of Neurophysiology 58, 251–266.
Vorobjev, V.S., 1991. Vibrodissociation of sliced mammalian nervous MacDonald, J.F., Bartlett, M.C., Mody, I., Pahapill, P., Reynolds, J.N., tissue. Journal of Neuroscience Methods 38, 145–150.
Salter, M.W., Schneiderman, J.H., Pennefather, P.S., 1991. Actions Vorobjev, V.S., Sharonova, I.N., 1994. Tetrahydroaminoacridine of ketamine, phencyclidine and MK-801 on NMDA receptor cur- blocks and prolongs NMDA receptor-mediated responses in a volt- rents in cultured mouse hippocampal neurones. The Journal of age-dependent manner. European Journal of Pharmacology 253, McKernan, R.M., Whiting, P.J., 1996. Which GABAA-receptor sub- Vorobjev, V.S., Sharonova, I.N., Haas, H.L., 1996. A simple perfusion types really occur in the brain? Trends in Neurosciences 19, system for patch-clamp studies. Journal of Neuroscience Methods Nayeem, N., Green, T.P., Martin, I.L., Barnard, E.A., 1994. Quaternary Wafford, K.A., Thompson, S.A., Thomas, D., Sikela, J., Wilcox, A.S., structure of the native GABAA receptor determined by electron Whiting, P.J., 1996. Functional characterization of human γamino- microscopic image analysis. Journal of Neurochemistry 62, 815– butyric acidA receptors containing the α subunit. Molecular Phar- Neher, E., 1983. The charge carried by single-channel currents of rat Woodhull, A.M., 1973. Ionic blockage of sodium channels in nerve.
cultured muscle cells in the presence of local anaesthetics. The Journal of General Physiology 61, 687–708.
Journal of Physiology (London) 339, 663–678.
Xu, M., Covey, D.F., Akabas, M.H., 1995. Interaction of picrotoxin Neher, E., Steinbach, J.H., 1978. Local anaesthetics transiently block with GABA receptor channel-lining residues probed in cysteine currents through single acetylcholine-receptor channels. The Jour- mutants. Biophysical Journal 69, 1858–1867.
nal of Physiology (London) 277, 153–176.
Zhorov, B.S., Bregestovski, P.D., 2000. Chloride channels of glycine Samoilova, M.V., Buldakova, S.L., Vorobjev, V.S., Sharonova, I.N., and GABA receptors with blockers: Monte Carlo minimization and Magazanik, L.G., 1999. The open channel blocking drug, IEM- structure–activity relationships. Biophysical Journal 78, 1786–

Source: http://www.synaptology.com/assets/files/articles/1.pdf