Introduction

Summary Introduction Materials & Methods Results Discussion References

Lanthanum is a trivalent cation and is the most electropositive element of the rare earth group. Its mechanisms of action at the cellular and molecular level have not been studied extensively until recently. Functional studies have indicated that lanthanum, unlike most divalent cations, increases GABA-activated currents in both native and recombinant GABARs (1-4). Based on earlier studies suggesting that changes in subunit composition produced recombinant receptors with differential selectivity toward benzodiazepines and the divalent cation zinc, we formulated this study to determine whether lanthanum displays similar subunit-dependent selectivity toward different GABAR isoforms. There is considerable interest in the effects of lanthanum and its compounds on cellular systems from a neurotoxicological point of view due to its increasing use in industry and therapeutics (5). There is evidence in cell culture and animal systems to suggest that micromolar concentrations of lanthanum can exert cytotoxic effects (6). Study of the cytotoxicity of lanthanum chloride in a pulmonary macrophage primary culture system indicated an half-maximum lethal concentration of 52 µM (7). Traces of lanthanum (0.5 µg/g) were detected in the bones of man and animals after exposure (8). More recent studies of autopsy specimens from deceased smelter factory workers exposed to lanthanum have indicated a 2- to 16-fold increase in lanthanum levels, compared with nonexposed controls; lanthanum levels were highest in those who died of lung cancer (5).

A number of different approaches have been taken to understand the mechanism of action of lanthanum and other heavy metals. Behavioral, anatomical, and biochemical approaches have been useful in identifying the overall toxic effects, structural changes in various regions of the nervous system and biochemical modifications caused by toxicants. However, functional studies using electrophysiological techniques could provide information about the cellular and molecular mechanisms of action of heavy metals and suggest how they alter the excitability of the nervous system and lead to behavioral changes (4). In the present study, we have compared and contrasted the effects of lanthanum on the electrophysiological properties of three different putative cerebellar GABAR isoforms transiently expressed in L929 fibroblasts using the whole-cell recording configuration. Specifically, we have examined the effects of changes in subunit subtype composition (among the three GABAR isoforms) on the nature and extent of modulation of GABAR currents by lanthanum. Our results demonstrate GABAR subunit-dependent actions of lanthanum.

	Materials and Methods

Summary Introduction Materials & Methods Results Discussion References

Plasmid construction. Full-length cDNAs encoding the rat 1, 3, and  GABAR subunits were kindly provided by Dr. A. J. Tobin (1; University of California, Los Angeles), Dr. D. B. Pritchett (3; University of Pennsylvania, Philadelphia, PA) and Dr. K. Angelides (; Baylor College of Medicine, Houston, TX) in the bluescript vector. The rat 2L and 6 subunits were cloned in our laboratory by Fang Tan (University of Michigan, Ann Arbor, MI). The rat cDNAs have been described previously (see Ref. 9 for review). The plasmids were cut with appropriate restriction enzymes to release the complete open reading frames and 10-100 bp of the 5 and 3 untranslated regions, including the Kozak sequences (10, 11). These plasmids were subcloned individually into the BglII site of the mammalian expression vector pCMVNeo (12) to form the plasmids pCMVr1, pCMVr6, pCMVr3, pCMVr2L, and pCMVr. A 3000 bp BglII fragment of pSV₂gal [obtained from Dr. Audrey Seasholtz (University of Michigan, Ann Arbor, MI) (13)] was subcloned into pCMVNeo to create the vector pCMVgal.

Preparation of gridded dishes. Individual 35-mm tissue culture dishes (Corning Glassworks, Corning, NY) were imprinted with a 26 × 26 grid (300 µm per grid edge) on the bottom with a Mecanex BB form 2 device (Medical Systems, Greenvale, NY) according to the manufacturer's instructions. After plating at low density, cells could be accurately located relative to a particular grid, identified by a corresponding two-letter alphabetic code, while switching between the fluorescent and the electrophysiology microscopes. The process of imprinting the grid removed some of the negative charges required for cell adherence, necessitating a coating of one or two drops of collagen (0.5 mg/ml) in PBS for optimal adherence of L929 cells. The gridded region of the dish was coated with collagen and UV-sterilized overnight before cells were plated on it.

Cell culture and DNA transfection. L929 cells were grown in Dulbecco's modified Eagle's medium with 10% horse serum along with 100 IU/ml of penicillin and 100 µg/ml of streptomycin at 37° in 5% CO₂/95% air. Cells were passaged the night before they were to be transfected with trypsin/EDTA solution (0.5% and 0.2%, respectively) and plated at 70% confluency (500,000 cells per 60-mm dish) in a 60-mm dish. The next day, cells were transfected with various combinations of CsCl-banded pCMVr1, pCMVr6, pCMVr3, pCMVr2L, pCMVr, and pCMVrgal plasmids, using a modified calcium phosphate precipitation method (14). Plasmids were mixed in a 1:1:1 (::gal) or 1:1:1:1 (:::gal or :::gal) ratio while maintaining the total amount of DNA added per dish at 16-20 µg in 500 µl of transfection buffer. Cells were shocked with a 15% glycerol/1 × PBS solution for 30 sec, 4 or 5 hr after the addition of precipitate. Cells were passaged as above 24 hr after addition of precipitate and placed in 15-ml conical tubes and treated with 375 µg/ml tissue culture grade DNase I for 5 min (twice, for a total time of treatment with DNase I of 10 min) at 37°. Cells were pelleted at 400 × g and plated onto either standard 35-mm plates or mecanex-gridded plates. Electrophysiological analysis was performed 24 hr later.

Galactosidase staining protocols. Two different -galactosidase staining protocols were used to identify cells transfected with pCMVgal. To determine the transfection efficiency, 5-bromo-4-chloro-3-indoyl -D-galactosidase staining of cells was performed as described previously (15). FDG staining was performed as originally described by Nolan et al. (16), with some modifications for use with adherent cells, to identify positively transfected cells for electrophysiological recordings. Cells were washed twice with PBS to remove the medium and incubated for 5 min at 37° with 1 ml of PBS to re-equilibrate the cells to this temperature. While the cells were incubating, 20 mM FDG solution prepared by the manufacturer (Molecular Probes, Eugene, OR) was diluted 1:20 by adding 25 µl of the 20 mM FDG solution into 500 µl of 0.5 × PBS in a 1.5-ml microcentrifuge tube and placed in a 37° water bath. After 5 min of incubation, PBS was aspirated from the cells, and the warmed 1-mM FDG solution (final concentration) was added to the cells. The plate with the cell and FDG solution was warmed in the 37° water bath for 1 min, placed on ice, and then 2.5 ml of ice-cold 1 × PBS was added. After 5 min on ice, the cells were viewed with a fluorescence microscope fitted with fluorescein filters.

Recording solutions and electrodes. Before recording, the PBS/FDG solution on the plate of cells was exchanged with five 2-ml washings of external recording medium containing the following: 142 mM NaCl, 8.1 mM KCl, 6 mM MgCl₂, 1 mM CaCl₂, 10 mM glucose, 10 mM HEPES, pH ~7.4. The internal (intrapipette) solution contained 153 mM KCl, 1 mM MgCl₂, 5 mM EGTA, 10 mM HEPES, pH ~7.3. This combination of external and intrapipette solutions produced a chloride equilibrium potential of 1.4 mV and a potassium equilibrium potential of 75 mV across the patch membrane. GABA was diluted with external recording solution from a stock solution (100 mM or 10 mM in distilled water) to the indicated final concentration on the day of the experiment. A multipuffer application system (50-90 µm tip diameter) was used to apply a range of different concentrations of drugs for experiments.

Multipuffer system to apply a range of different concentrations of drugs. To enable fast application of a number of different concentrations and types of drug, a multipuffer application system was designed in the laboratory (17). Briefly, it consisted of a T-tube device with inlet and outlet ports feeding into a common application port at one end and individually connected to polyethylene tubing leading to either a reservoir of different solutions to be tested (inlet tubing) or the waste flask (outlet tubing) at the other end. Puffer tips between 50 and 90 µm in diameter made of nonfilament glass were inserted into the application port. A suction pump (aquarium air pump; Supra, Oakland, NJ) was connected to the outlet tubing of the U-tube device via a three-way miniature solenoid valve (General Valve, Fairfield, NJ) operated by a valve driver (Valve driver II; General Valve). To apply a drug, the valve was turned off (regulated by timer), stopping the suction of solution through the U-tube device and pushing the resultant column of accumulated solution in the application port out through the puffer tip. Reactivation of the valve resumed flow of solution through the U tube and suction of the applied drug/solution from the bath, thus affecting a washout of the drug from the area around the puffer tip and cell. The multipuffer application system was tested for a satisfactory rate of application and removal of drug from the bath before every experiment using the dye, fast green (Sigma Chemical, St. Louis, MO) in a petri dish filled with distilled water. The rate of application and removal of the solutions depended on the size of the tip and its position relative to the cell [ between 30 and 70 msec, measuring tip potential between potassium-free (0 mM KCl) and potassium-containing (120 mM KCl) solutions].

Whole-cell recordings and analyses. Whole-cell recording was performed with methods described previously for mouse spinal cord neuron recordings (18, 19) using a List L/M EPC-7 amplifier (List Electronics, Darmstadt, Germany). All recordings were made at room temperature (22-24°). Currents were recorded simultaneously on a video cassette recorder (Sony SL-HF360; Sony, Tokyo, Japan) via a digital audio processor (Sony PCM-501 ES, 14-bit, 44 kHz), on Axotape (Version 2; Axon Instruments, Burlingame, CA; using an Axon TL-1-40 16-channel, 40-kHz, 12-bit interface) on a IBM-compatible 80286 personal computer and a chart recorder (Gould, Cleveland, OH) for later computer analysis. Whole-cell recordings were low-pass filtered (3 db at 1 kHz, 8-pole Bessel filter; Frequency Devices, Haverhill, MA) before the chart recorder. The peak whole-cell current amplitudes were measured either using Axotape or directly from the chart output and reported as mean ± standard error. Statistical tests of significance were performed using paired Student's t test for all drug treatments and the p values were reported. Concentration response curves were fitted to a four-parameter logistic function R = R_min + (R_max  R_min)/(1 + 10)^{Log EC_{50 X)n),}} where X is logarithm of drug concentration, R is the response to drug, R_max is the maximum drug response, R_min is the minimum drug response, Log EC₅₀ = X value when the response is halfway between maximum and minimum, and n is the Hill slope, a unitless variable that controls the slope of the curve (Prism; GraphPAD Software, San Diego, CA).

	Results

Summary Introduction Materials & Methods Results Discussion References

GABA responsiveness of transfected L929 cells. Previous work from our laboratory has shown that six putative cerebellar GABAR isoforms, 132L, 122L, 632L, 622L, 63, and 12L GABARs, show high levels of functional expression after transient transfection in mouse fibroblast L929 cells (20). Concentration response curves for GABA corresponding to 132L, 632L, and 63 GABAR isoforms showed typical sigmoidal shapes with different EC₅₀ values for GABA and maximal GABA-evoked current amplitudes. The 63 GABARs displayed an EC₅₀ value of 0.3 µM and a maximum current amplitude of 371 ± 116 pA (eight experiments), the 632L GABARs showed an EC₅₀ value of 2 µM and a maximum current amplitude of 730 ± 305 pA (nine experiments), and the 132L GABARs exhibited an EC₅₀ value of 14 µM and a maximum current amplitude of 803 ± 141 pA (nine experiments) (20).

Modulation of GABAR currents by the polyvalent cation lanthanum. To determine the effect of lanthanum on the three putative cerebellar GABAR isoforms, we examined the effect of 300 µM lanthanum on GABA-evoked 63, 632L, and 132L whole-cell currents at submaximal concentrations of GABA (close to the respective EC₅₀ values for GABA). The concentrations used were 0.3, 3, and 10 µM GABA for the 63, 632L, and 132L GABAR isoforms, respectively (Fig. 1). 300 µM lanthanum enhanced 10 µM GABA-evoked 132L currents to 145 ± 12% (mean ± standard error, n = 8) of the control current evoked by GABA alone. In contrast, it did not potentiate 632L and 63 GABAR currents, blocking 632L currents by 30 ± 10% (mean ± standard error, n = 8) and 63 currents by 81 ± 5% (mean ± standard error, n = 8) (Fig. 2).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of 300 µM lanthanum on GABA-evoked whole-cell currents recorded from L929 cells transfected with 132L, 632L, and 63 GABAR subtypes. Cells were voltage-clamped at 75 mV, and 5-sec pulses of submaximal concentrations of GABA with and without 300 µM lanthanum were applied via pressure-ejection micropipette placed close to the cells. Downward deflections, inward currents (efflux of negatively charged chloride ions) at 75 mV (ECl = 0 mV). Horizontal bars above the current traces, the duration of application of GABA. The recovery of current amplitudes after washout of lanthanum to pretreatment values indicates reversibility of the effect of lanthanum.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Normalized concentration-response curves for lanthanum corresponding to cells transfected with 132L (A), 632L (B), and 63 (C) GABAR subtypes. Symbols, mean values of current amplitudes as a percent of maximum current induced by 10, 3, and 0.3 µM GABA for the 132L (n = 4), 632L (n = 8), and 63 (n = 8) GABAR isoforms, respectively, in the absence of lanthanum. Vertical bars, mean ± standard error.

Competitive interaction between lanthanum ions and GABAR isoforms. To elucidate the mechanism of potentiation and inhibition of different GABAR isoforms by lanthanum, GABA concentration response curves were compared in the absence and presence of the corresponding EC₅₀ and IC₅₀ values of lanthanum for each of the three GABAR isoforms. The current amplitudes in the absence and presence of lanthanum were normalized to the maximum current evoked by GABA in the absence of lanthanum. The data were fitted by the logistic equation described in Materials and Methods. In the presence of 300 µM lanthanum, the concentration response curve for 132L GABARs was shifted to lower concentrations of GABA with no increase in the maximum current evoked by GABA (Fig. 3A). The EC₅₀ value for GABA in the absence of lanthanum was 13 ± 3.3 µM and the Hill slope was 1.5 ± 0.1 (n = 4). In the presence of 300 µM lanthanum, the EC₅₀ value for GABA was decreased to 6.4 ± 3 µM and the Hill slope was not significantly changed at 2.0 ± 0.2 (n = 4). The decrease in EC₅₀ value for GABA was statistically significant (p = 0.01) using paired Student's t test, and the Hill slope was not changed significantly (p = 0.18). Therefore, lanthanum decreased the GABA EC₅₀ value for 132L GABARs without changing the maximum current, suggesting that lanthanum increased the affinity of GABA for the 132L GABARs without changing the number of functional receptors. Unlike the 132L GABAR isoform, concentration response curves for the 632L and 63 GABAR isoforms were shifted to higher concentrations of GABA in the presence of lanthanum with no decrease in the maximum GABA-evoked current. The increases in EC₅₀ value for GABA were statistically significant (p < 0.05). The EC₅₀ value for GABA was increased from 2.5 ± 0.2 µM to 4.4 ± 0.1 µM GABA in the presence of 100 µM lanthanum (p = 0.004, n = 4) for the 632L GABAR isoform (Fig. 3B), and the EC₅₀ value for 63 GABAR isoform was increased from 0.38 ± 0.05 µM to 1.06 ± 0.23 µM GABA in the presence of 30 µM lanthanum (p = 0.04, n = 5) (Fig. 3C). The Hill slope for 632L GABAR currents was decreased from 1.48 ± 0.09 to 1.22 ± 0.09 (p = 0.003, n = 4), and that for 63 GABAR currents was altered from 1.22 ± 0.15 to 1.15 ± 0.17 (p = 0.8, n = 5). Therefore, lanthanum decreased the GABA affinity for the 632L and 63 GABAR isoforms without altering the number of active receptors.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Competition curves for GABA and lanthanum for 132L, 632L, and 63 GABAR isoforms. Concentration response curves for GABA were obtained in the absence and presence of 300 µM lanthanum (132L; A), 100 µM lanthanum (632L; B), and 30 µM lanthanum (63; C). Symbols, mean values (n = 3) of currents normalized to the maximum current evoked in the absence of lanthanum from three cells each for each of the three different isoforms. Vertical bars, mean ± standard error.

Voltage independence of the effect of lanthanum on different GABAR isoforms. To gain some insight regarding the location of the site of interaction between lanthanum and GABAR chloride channel, the voltage dependence of GABAR currents was examined in the presence and absence of lanthanum (Fig. 4). The current-voltage relationship in the presence of GABA alone was largely linear for the 132L and 632L isoforms (Fig. 4, A and B) with reversal potentials of 0 and 6 mV, respectively, and it showed some departure from linearity for the 63 isoform with a reversal potential of +5 mV (Fig. 4C). The average values of the reversal potentials for the three isoforms in the absence and presence of lanthanum were 4.3 ± 2.4 and 4.9 ± 1.4 mV (132L, n = 6, p = 0.7), 2.0 ± 2.4 and 2.5 ± 2.0 mV (632L, n = 6, p = 0.8) and 3.8 ± 0.3 and 5.7 ± 2.5 mV (63, n = 5, p = 0.5), respectively. There was no change in the current-voltage relationship in the presence of lanthanum for any of the three GABAR isoforms studied, consistent with voltage-independent potentiation and inhibition by lanthanum.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Current-voltage relationships for 132L, 632L, and 63 GABAR isoforms in the absence and presence of lanthanum. Representative current-voltage curves for GABA currents evoked in the absence and presence of lanthanum are shown for 132L (10 µM GABA+300 µM lanthanum; A), 632L (3 µM GABA+100 µM lanthanum; B), and 63 (0.3 µM GABA+30 µM lanthanum; C) GABAR isoforms. Step changes in holding voltage were applied for voltages ranging from 75 mV to +60 mV.

	Discussion

Summary Introduction Materials & Methods Results Discussion References

Subunit-dependent inhibition and potentiation of different GABAR isoforms by lanthanum. In contrast to the mainly inhibitory effects of divalent cations, the trivalent cation lanthanum has been shown to enhance GABAR currents in rat DRG neurons (2) and in recombinant 12 and 122 GABARs expressed in human embryonic kidney (A293) cells (3). Reichling and MacDermott (1) reported a biphasic effect of lanthanum on rat dorsal horn neurons: at concentrations between 1 and 100 µM, lanthanum-enhanced GABAR currents to a maximum of 130% of control, whereas at higher concentrations, lanthanum markedly reduced GABAR currents (1).

Extracellular location of site for lanthanum interaction. Results from the present study suggest that the site of interaction with lanthanum lies on the extracellular surface of GABARs. First, the quick onset and removal of the effect of lanthanum during acute application (5-sec pulses) of lanthanum in the whole-cell recording configuration suggests that the action is mediated by an extracellular event. Second, the competitive nature of potentiation and inhibition by lanthanum with respect to GABA also suggests that the site of interaction between GABARs and lanthanum is likely to be extracellular. Third, the lack of a clear voltage dependence in the current-voltage relationship in the presence of lanthanum as compared with that in its absence also suggests that the site for lanthanum interaction sensed little or none of the transmembrane electrical gradient and, therefore, was likely to be extracellular. Earlier work by Ma and Narahashi (2) also suggested that the potentiation by lanthanum was nearly voltage independent, whereas that of Im et al. (3) suggested weak voltage dependence. Based on our results, the extracellular lanthanum interaction site on GABAR is subject to characteristic functional modulation by the -subunit subtype (potentiation versus inhibition) and the / subunits (extent of inhibition) and, therefore, might be composed mainly of contributions from the extracellular regions of at least the  and / subunits.

Physiological implications of enhancement and inhibition of distinct GABAR isoforms by lanthanum. Potentiation of the 1-containing and inhibition of the 6-containing (putative) cerebellar GABAR isoforms by lanthanum might increase the plasticity of the cerebellum from a therapeutic or functional perspective. GABAergic neurotransmission has been proposed to play an important role in the perception of pain. Selective depression of noxiously evoked activity from rat and feline spinal cord neurons by intravenous injection of midazolam and clinical control of pain by subarachnoid infusion of midazolam have been reported (28-30). It has been postulated that pain perception is modulated in the substantia gelatinosa (lamina II), which controls impulse transmission from the primary afferents to projecting neurons (31, 32). By demonstrating a striking segregation of distinct GABAR subunits in functionally different neuron populations in the substantia gelatinosa (2, 3) and in projecting neurons (1), Bohlhalter et al. (26) have suggested differential modulation of GABAergic neurotransmission by specific pharmacological agents and pharmacological control of nociception and treatment of neurogenic pain based on GABA_A receptor heterogeneity in the future. Lanthanum administered into the lumbar subarachnoid space of rat has been shown to have antinociceptive effects (33). Based on the subunit-specific modulation of GABAR currents by lanthanum described in the present study, lanthanum could be an important resource in the functional characterization of GABAR isoforms in the substantia gelatinosa and projecting neurons ultimately providing insights into the role of receptor heterogeneity in signal processing and mechanisms of pain.