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Original Article |
suchyna{at}acsu.buffalo.edu
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ABSTRACT |
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45 pS at –100 mV) and a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appeared specific since it had no effect on whole-cell voltage-sensitive currents. The equilibrium dissociation constant of 630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces 40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cell size. This is the first report of a peptide toxin that specifically blocks stretch-activated currents. The toxin affect on swelling-activated whole-cell currents implicates SACs in volume regulation.
Key Words: mechanogated • swell • astrocyte • ventricular • myocytes
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INTRODUCTION |
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TopABSTRACTINTRODUCTION METHODS RESULTS DISCUSSION REFERENCES |
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), K + delayed rectifier, voltage-gated Na+ ( Elinder and Arhem 1994), and Ca2+ ER release channels (Kluesener et al. 1995). Also, a variety of "specific" blockers for voltage- and ligand-gated channels (e.g., amiloride, cationic antiobiotics, tetrodotoxin, tetraethylammonium, quinidine, diltiazem, and verapamil) exhibit low affinity blocking activity against SACs (for review, see Hamill and McBride 1996; Sachs and Morris 1998).
SACs have been implicated as either activators or modifiers of many different cellular responses to mechanical stimuli, including modification of electrical and contractile activity of muscle tissue, involvement in volume regulatory ion fluxes, and initiation of action potentials in specialized sensory cells such as inner hair cells of the cochlea and Merkel cells in the epithelium (Sachs 1992; Sachs and Morris 1998; Tazaki and Suzuki 1998). However, it has proven difficult to definitively associate mechanically stimulated physiological responses with specific SACs in the absence of an inhibitor.
This limitation is nowhere more evident than in volume regulation studies, where cells undergo a regulated volume decrease (RVD) in response to hypotonic stress. RVD is produced by efflux of cytoplasmic inorganic osmolytes (mainly K+ and Cl–) and small organic molecules. K+ and Cl– efflux occurs via cotransporters and individual conductive channels that are separate, but interdependent (for reviews, see Chamberlin and Strange 1989; Hoffmann and Simonsen 1989; Sarkadi and Parker 1991; Pasantes-Morales 1996). RVD in astrocytes has been intensely studied due to its importance in controlling brain edema ( Kimelberg 1995; Pasantes-Morales 1996). These cells display a fast RVD response and possess a high resting K+ flux. Within the first minute after hypotonic swelling of neonatal astrocytes (160 mOsM reduction), the membrane potential depolarizes by 50 mV (Kimelberg et al. 1990). The depolarization is primarily caused by a rapidly activated 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS)–sensitive anion current ( Pasantes-Morales et al. 1994; Bakhramov et al. 1995). There is also a Ca2+ influx during hyposmotic swelling that is partially mediated by opening of dihydropyridine-sensitive Ca2+ channels ( O'Connor and Kimelberg 1993; Bender et al. 1994). It is important to remember that most agents purported to be specific, such as dihydropyridines, have not been tested against SACs.
A number of studies have suggested that nonselective cation-permeable SACs play a role in both membrane depolarization ( Kimelberg and Kettenmann 1990) and Ca2+ influx observed during RVD (Christensen 1987; O'Connor and Kimelberg 1993; Chen et al. 1996). In addition, K+-selective SACs and a curvature-sensitive nonselective cation channel have been described in neonatal astrocyte cultures (Bowman et al. 1992; Islas et al. 1993). A cation-selective SAC has also been identified in C6 glioma cells (Bowman and Lohr 1996). However, none of these channels have been investigated for involvement in RVD.
SACs have also been implicated in mechanical sensitivity of the heart. Mechanical stimulation of cardiac myocytes and whole heart preparations can cause depolarization, extrasystoles, and arrhythmias (see Hu and Sachs 1997). Also, chronic hemodynamic stress that leads to congestive heart failure (CHF) and the accompanying cellular hypertrophy may be initiated by stretch- or swelling-activated currents ( Sachs 1988; Vandenberg et al. 1996; Clemo and Baumgarten 1997). CHF chronically activates a whole-cell cation-selective current previously identified with hypotonic swelling or chronic rapid pacing (Clemo et al. 1998). A blocker of SACs could have clinical use.
Chen et al. 1996 showed that crude Grammostola venom could block SACs in an outside-out patch from GH3 pituitary cells. They also demonstrated the venom can block Ca2+ uptake during hypotonic swelling, but not during high K+ depolarization, which would activate voltage-gated Ca2+ channels. Thus, it was suggested that Ca2+ uptake was triggered by the activation of SACs. The primary aim of this study was to isolate and characterize the active component from Grammostola venom.
To isolate this SAC-blocking component(s), fractions of the venom were screened by perfusion onto outside-out patches from adult rat astrocytes, a preparation in which SACs could be maintained active. A single component peak was identified and sequenced, revealing a unique peptide (noted GsMTx-4) containing an inhibitor cysteine knot (ICK) consensus motif ( Narasimhan et al. 1994). The toxin exhibited negligible activity against voltage-sensitive whole-cell currents. However, the toxin did reduce swelling-activated whole-cell currents in astrocytes and CHF model cardiac myocytes. The effect of this new toxin on whole-cell currents, for the first time, directly implicates specific cation-selective SACs in the response to swelling.
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METHODS |
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), but Grammostola is used here to maintain consistency with earlier biomedical publications. Venom was collected by an electrical milking procedure ( Bascur et al. 1982) and stored at –80°C. It was fractionated by high-performance liquid chromatography, incorporating Beckman System Gold 126 solvent delivery and 168 photodiode-array detector modules (Beckman Instruments, Inc.), and using linear gradients with a flow rate of 3.5 ml/min unless noted. Whole venom (825 µl) was separated into eleven 75-µl aliquots that were each diluted to 2 ml each with 15% solvent B. Solvent A was 0.1% trifluoroacetic acid (TFA) in water and solvent B was 0.1% TFA in acetonitrile. The diluted venom was fractionated on a Zorbax RX-C8 (9.4 x 250 mm, 5 µm, 300 Å; Mac-Mod Analytical, Inc.) reversed-phase (RP) column equilibrated in 15% solvent B. The column was developed with a 40-min gradient (15–55% solvent B) begun 3 min after injection of the sample with a flow of 3.5 ml/min. The effluent was monitored at 280 nm and fractions were collected as noted on the chromatogram (see Fig. 3 A). Similar fractions from all 11 chromatographies were combined, lyophilized, and tested for bioactivity. The assay samples were dissolved in 140 mM NaCl, 10 mM HEPES, 5 mM KCl, 2 mM MgSO4 , to a final dilution of 1:1,000, relative to whole venom, for testing on outside-out patches. Several of the pools showed partial block of the SACs (as described below), but only pool 9 gave consistent, complete block of the channels.
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pH 11 from the MonoS column with a gradient from pH 7–12 (50 mM sodium phosphate buffer, 0.1 M in NaCl), but no resolution from other components was visible.
Therefore, pool B was subjected to a final RP chromatography to remove a small amount of earlier and later eluting peptides. Pool B was diluted to 4 ml with 20% solvent B and 0.5-ml portions chromatographed on the Zorbax column described above, eluting with a 7-min gradient (20–27% solution B), followed by a 46-min gradient (27–50% solution B), and the effluent was monitored at 220 nm (see Fig. 3 C). The first gradient was begun 5 min after injection of the sample. The active peptide, GsMTx-4, eluted between 29.5 and 30.5 min. Corresponding fractions from the eight chromatographies were pooled to give 7.5 mg of GsMTx-4. The average yield of GsMtx-4 from several purifications was 8 mg/ml of venom fractionated, which implies that the toxin is 2 mM in whole venom. The purity of the final product used in single channel and whole cell assays was assessed by analytical chromatography on an Aquapore RP300 C8 column (4.6 x 220 mm, 7 µm, 300 Å; PE Biosystems), eluting with a 25-min linear gradient (32–47% solution B) with a flow of 1 ml/min monitored at 220 nm (see Fig. 3 D). Elution with a gradient of methanol/water (0.1% in TFA) gave a similar profile with a longer retention time, but revealed no other impurities.
Mass Spectrometry
1 µl of the sample solutions (intact toxin or fragments) in 0.1% TFA (or the HPLC elution solvent) were mixed on the sample plate with 1 µl of a saturated solution of 4-hydroxy-
-cyanocinnamic acid in 1:1 CH 3CN:0.1% aqueous TFA. The solution was allowed to air dry before being introduced into the mass spectrometer. Spectra were acquired on a PerSeptive Biosystems Voyager Elite MALDI-TOF (matrix-assisted laser desorption ionization–time of flight) instrument operated in linear delayed extraction mode (50–100 ns). The instrument was equipped with a nitrogen laser (3-ns pulse). The acceleration potential was 22 kV.
Sequencing
The toxin was further purified by microbore RP-HPLC (0.8 x 250 mm C18 column, with a linear gradient from 0.1% TFA-15% CH3CN to 0.1% TFA-70% CH3CN in 90 min, flow rate 40 µl/min, monitored at 214 nm). The toxin peak was collected at 24.6 min. The HPLC fraction (1 nmol) was dried down and taken up in 80 µl 8-M guanidine HCL-100 mM Tris-5 mM tributylphosphine, pH 8.5, and incubated for 8 h at 55°C. N-Isopropyliodoactamide (1 mg in 20 µl MeOH + 80 µl Tris) was added and the solution was incubated for an additional 2 h at room temperature. The reduced and alkylated peptide was then desalted by HPLC on a C18 column as described above (elution time, 30.1 min). NH2-terminal sequencing was carried out on an ABI 477 after loading the reduced and alkylated peptide on PVDF membrane.
Digestion with BNPS [(2-2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine]-skatole ( Fontana 1972) was carried out by dissolving the purified reduced and alkylated peptide in 50 µl 0.1% TFA and 15 µl BNPS-skatole. The solution was incubated at room temperature for 8 h. The digestion products were separated by HPLC as described above. Two main peaks were collected and sequenced by Edman degradation. Asp-N digestion ( Wilson 1989) was performed by dissolving the purified reduced and alkylated peptide in 100 mM Tris, pH 8.0 and treating with 1% (wt/wt) Asp-N for 20 h at 35°C. The fragments were separated and analyzed by mass spectrometry before Edman degradation.
Astrocyte Cell Culture
Activated adult astrocytes, isolated from gelatin-sponge implants from adult Sprague-Dawley rat brains (Langan et al. 1995), were provided courtesy of Dr. Thomas Langan (SUNY Buffalo, Buffalo, NY) at passages 2–4. Astrocytes were maintained in DMEM, 10% fetal bovine serum, and 1% penicillin/streptomycin and were used in experiments between 2 and 5 d after passage. Cells between passages 4 and 35 expressed SACs with the same properties. Both stellate and polygonal (fibroblast-like) cells were used.
Astrocyte Single-Channel Patch Clamp
Patch voltage was controlled by an Axopatch 200B (Axon Instruments) and stored directly on computer disk via a Labmaster DMA version B (Scientific Instruments) board controlled by pClamp6-Clampex acquisition software (Axon Instruments). Currents were sampled at 10 kHz and low-pass filtered at 2 kHz through a four-pole Bessel filter on the Axopatch 200B. Experimental voltage protocols were controlled by pClamp6-Clampex. All potentials are defined with respect to the extracellular surface.
Electrodes were pulled on a pipette puller (PC-84; Brown-Flaming Instruments), painted with Sylgard 184 (Dow Corning Corp.) and fire polished. Electrodes were filled with KCl saline containing (mM): 140 KCl, 5 EGTA, 2 MgSO4, 10 HEPES, pH 7.3) and had resistances ranging from 3 to 8 M
. Bath saline consisted of (mM): 140 NaCl, 5 KCl, 1 MgSO4, 1 CaCl2, 6 glucose, and 10 HEPES, pH 7.3.
Pressure and suction were applied to the pipette by a pressure clamp designed and constructed in our laboratory by Dr. Steven Besch. Pressure values refer to pressure in the pipette; i.e., the intracellular side of the membrane in outside-out patches. Suction applied to a cell-attached patch has the same sign as pressure applied to an outside-out patch. The rise time of pressure changes at the tip were determined by monitoring the rate of current change when pressure steps were applied to an electrode containing 150 mM KCl solution and placed in a water bath. The 
10–90 was 5 ms, as determined by exponential fits to the current decay. Perfusion of the patch was handled by a pressurized bath perfusion system with eight separate channels (BPS-8; ALA Scientific).
Offline data analysis was performed with pClamp6 analysis software and Origin 5.0. Maximal unitary channel currents were determined via Gaussian fits to the peaks of the all-points amplitude histograms from records containing one to three channels. Many current records displayed more than three channel openings (maximal single channel currents plus subconductance states) and were impossible to fit using Pstat software. Some of these records were analyzed by determining all step-like changes in the current during the pressure application and selecting the average maximal current level as the unitary current. The data analyzed by this method was in good agreement with the unitary current levels determined by analysis of all points amplitude histograms from single-channel patches.
Astrocyte Whole-Cell Current Clamp
Whole-cell current was measured by the Nystatin-perforated patch technique (Horn and Marty 1988). Bath saline was the same as above. Pipette saline consisted of (mM): 80 KCl, 30 K2SO4 , 10 NaCl, 3 MgSO4, 0.13 CaCl2, 0.23 EGTA, and 10 HEPES, pH 7.3. Nystatin was dissolved in pipette saline to a final concentration of 200 µg/ml. After patch formation, access resistance was allowed to drop to 15 M (uncompensated), after which the series resistance compensation was set at 65%, and prediction was set to 75%. Whole-cell capacitance measurements ranged from 25 to 50 pF. Whole-cell currents were monitored by either a voltage-step protocol (see Fig. 9), or by 600-ms voltage ramps. During hypotonic swelling, the cell was perfused initially with isotonic saline (bath saline with 160 mM mannitol replacing 80 mM NaCl) before switching to hypotonic saline (isotonic saline minus 140 mM mannitol). The BPS-8 perfusion system described above was used to rapidly (<200 ms) change the bathing solution. Peak currents were measured at 3–5 ms into voltage steps.
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) using a collagenase–pronase dispersion method (Clemo and Baumgarten 1997). Cells were stored in a modified Kraft-Bruhe solution containing (mM): 132 KOH, 120 glutamic acid, 2.5 KCl, 10 KH2PO4, 1.8 MgSO4 , 0.5 K2EGTA, 11 glucose, 10 taurine, 10 HEPES, pH 7.2. Myocytes were used within 6 h of harvesting and only quiescent cells with no evidence of membrane blebbing were selected for study.
Cardiac Myocyte Electrophysiology and Volume Determination
For a detailed explanation of methods, see Clemo and Baumgarten 1997. In brief, electrodes were pulled from glass capillaries to give a final tip diameter of 3–4 µm and a resistance of 0.5–1 M when filled with the standard electrode filling solution containing (mM): 120 K aspartate, 10 KCl, 10 NaCl, 3 MgSO4, 10 HEPES, pH 7.1. Whole-cell currents were recorded using an Axoclamp 200A. Pulse and ramp protocols, voltage-clamp data acquisition, and offline data analysis were controlled with custom programs written in ASYST. Both step and ramp voltage-clamp protocols were applied with a holding potential of –80 mV. Currents were digitized at 1 kHz and low-pass filtered at 200 Hz. Whole-cell currents were recorded using the amphotericin perforated-patch technique. Solution changes were performed by bath perfusion that was completed within 10 s. The standard bath solution contained (mM): 65 NaCl, 5 KCl, 2.5 CaSO4 , 0.5 MgSO4, 10 glucose, and 10 HEPES, pH 7.2, and 130 (1T) or 283 (1.5T) mannitol to control the osmolarity. Isotonic osmolarity was taken as 296 (1T) and 444 (1.5T) mosm for hypertonic solution. Myocyte volume was determined by visualization with an inverted microscope (Diaphot; Nikon Inc.) equipped with Hoffman modulation optics and a high-resolution TV camera coupled to a video frame grabber. Images were captured online each time a ramp or step voltage-clamp protocol was performed using a program written in C and assembler and linked to ASYST voltage-clamp software. A combination of commercial (MOCHA; SPSS Inc.) and custom (ASYST) programs were used to determine cell width, length, and area of the image.
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RESULTS |
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; Bowman and Lohr 1996; Islas et al. 1993) due to difficulties in culturing adult astrocytes. Langan et al. 1995 showed that adult-activated rat astrocytes from gel foam implants could be isolated and cultured through >10 passages, while maintaining all type 1 astrocytic markers [glial fibrillary acidic protein (GFAP), vimentin, 7B11, and RAN2], contact inhibition, and cell cycle kinetics. In cell-attached patches, activated adult astrocytes express primarily one type of SAC that can be activated by both pressure and suction (Fig. 1 A, only pressure data shown). Observation from >100 patches typically showed two to five channels/patch. Consistent with the results of Hamil and McBride (1992) and Small and Morris 1995, SAC activity in cell-attached patches was sensitive to the level of suction used in seal formation. Channels were rarely observed when >10 mmHg of suction was used during seal formation, whereas >90% of patches showed channel activity with <10 mmHg. With 140 mM KCl in the electrode, the single-channel conductance inwardly rectified being 46 pS at –100 mV, but only 21 pS at +100 mV (Fig. 1 C). Channel activity was normally initiated by applying between 25 and 35 mmHg of suction. However, rundown did occur so that increasing levels of suction were required to activate the channels over the 5–10 min during which data was acquired.
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). The time dependence of P o can be described by an initial phasic period followed by a tonic period, as defined in Bowman and Lohr 1996. Both the duration of the phasic period and Po during the tonic period showed a steep voltage dependence, decreasing as the potential became more negative (Fig. 1 A, see average currents). Of 16 cell-attached patches analyzed, 12 displayed adaptation at hyperpolarized potentials. In addition to adaptation, multiple voltage-dependent substates were visible at –100 mV, compared with only one at depolarizing potentials. Kinetic analysis of these substates is currently in progress. One major subconductance state observed at positive potentials (not shown in this figure) was 80–90% of the maximal conductance state, similar to that reported in Xenopus oocytes (Silberberg and Magleby 1997). Adaptation was also observed in C6 glioma cells (Bowman and Lohr 1996). However, in contrast to the rapid adaptation observed in adult astrocytes, adaptation in C6 cells occurred over 1–2 s and was not as strongly voltage dependent.
Channel activity in outside-out patches was generally similar to that in cell-attached patches, but they had different adaptation properties ( Fig. 1 B). The SACs opened in response to both pressure and suction. With 140 mM KCl in both the pipette and the bath, the I-V profile (44 pS at –100 mV, and 21 pS at +100 mV, cytoplasmic side) was nearly identical to that observed for cell-attached patches (Fig. 1 D). In this configuration, the channels were initially activated by between 30 and 40 mmHg of pressure. The similarities between the conductance and pressure sensitivity in the two patch configurations suggest that these channel properties have not been significantly modified by outside-out patch formation. However, of 12 outside-out patches, only one displayed the fast adaptation property observed in cell-attached patches. Instead, two showed no change in Po with respect to time or voltage, while the remaining nine patches exhibited a slow increase in current at both positive and negative voltages, where the number of active channels increased during the 500-ms pressure step (Fig. 1 B, 100 mV, and see Fig. 5 A, average control current). The rate of increase was greater for pressure steps at positive voltages due to an increase in Po at positive potentials. Similar responses are observed in Xenopus oocytes when large pressure stimuli are applied to eliminate the adaptation property (see Figure 2 in Hamill and McBride 1992). Outside-out formation is likely to disrupt the associations between membrane and cytoplasmic attachments in a similar way to the high pressure stimuli applied to cell-attached patches of the Xenopus oocyte.
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). Like other nonselective SACs, the astrocyte channels were not blocked by 130 mM Cs+ (data not shown). The channels were cation selective based on external ion substitution experiments with outside-out patches. When gluconate ( Fig. 2, 
) was substituted for Cl – (
), we saw no change in the I-V profile. However, substituting NMDG+ (
) for K+ in the bath produced an 88% reduction in current at –100 mV. The channel displayed a weak selectivity for K+ over Na+ since the current was reduced 24% at –100 mV when Na+ was substituted for K+ in the bath (Fig. 2 , 
). The properties of this SAC in adult astrocytes were similar to the cation-selective SACs described for many other cell types, including C6 glioma cells. SACs in C6 cells display a unitary conductance of 40 pS in equimolar 100-mM KCl are inwardly rectifying and show voltage-dependent adaptation (Bowman and Lohr 1996). However, SACs in our adult astrocyte differed from the highly K+ -selective channels (Islas et al. 1993) and from the 50-pS curvature-sensitive channels (Bowman et al. 1992) reported in neonatal astrocytes preparations.9
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; Lane et al. 1991), had no effect at 20 µM.
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). In neonatal astrocytes, the potent (nanomolar affinity) NMDA open-channel blocker MK801 inhibits glutamate-stimulated K+ influx ( Bender et al. 1998). Hence, we tested glutamate and MK801 on the adult astrocyte SACs. Glutamate at 10 µM did not activate SACs in unstretched patches that contained SACs, and 20 µM MK801 did not block SAC opening when stretched.
Some reports indicate that L-type Ca2+ channels are stretch sensitive (Langton 1993; Ben-Tabou et al. 1994) and that blockers of these channels are active against SACs (Ruknudin et al. 1993; Small and Morris 1995). Neither 100 µM diltiazem nor 50 µM nifedipine showed any blocking activity against the SACs in adult astrocytes. The anion channel blocker DIDS produced a significant reduction in the amount of swelling-activated anion current in astrocytoma cells (Bakhramov et al. 1995), but 50 µM DIDS had no effect on stretch-activated currents in the patch.
Identification and Characterization of a SAC Blocking Toxin from Grammostola Venom
HPLC fractions of Grammostola (Gs) were lyophilized, redissolved at a 1:1,000 dilution and perfused onto outside-out patches. Fraction 9, on the Gs whole-venom chromatogram (Fig. 3 A) blocked the SACs. This fraction was further resolved on two slower gradients (Fig. 3B and Fig. C) at 0.5% change in acetonitrile/min until a single peptide peak was identified containing the activity (Fig. 3 D). The amount of isolated peptide was determined by weight and three different protein spectroscopic methods, showing that a 1:1,000 dilution corresponds to a concentration of 8 µg/ml whole venom. The peak was determined to have a molecular weight of 4,093.90 by mass spectrometry and designated GsMTx-4 (for Grammostola mechanotoxin #4). Other peptides have been isolated from Gs venom that are active against SACs (Sachs, F., unpublished observations, and GsMTx-1 U.S. Patent #5756663); however, GsMTx-4 showed the most consistent and potent activity. At this concentration, the block was complete, and occurred rapidly upon superfusion of the patch, as shown by representative current traces in Fig. 4.
The association rate of the toxin was determined by applying toxin to an outside-out patch while the channels were activated by stretch. In the absence of GsMtx-4, channel activity increased over time at constant pressure (compare Fig. 1 B with 5 A). When 5 µM toxin was perfused onto the patch, 1 s after the initiation of the pressure step, the current decayed exponentially (Fig. 5 A, GsMTx-4). When the control and GsMTx-4 average current records are superimposed, before GsMTx-4 application, the currents are nearly identical (Fig. 5 B). The difference current was calculated (Fig. 5 C), and the period of GsMTx-4 application was fitted with a single exponential (Fig. 5 D), yielding a time constant of 594 ± 10 ms. Assuming a 1:1 binding, this gives an association constant, kA, of 3.4 x 105 M –1 s–1.
To determine the dissociation rate, we fit the increase in average patch current (n = 7 patches) during toxin washout. Fit to a single exponential, the washout time constant was 4.7 ± 1.7 s ( Fig. 6 B). From this dissociation constant ( kd = 0.21 s–1) and the association constant determined above (ka = 3.3 x 105 M–1 s–1), the calculated equilibrium constant, Kd = kd/ ka = 631 ± 240 nM (standard error calculated from the first-order approximation using the errors of ka and kd). Using the ratio of rate constants to evaluate Kd minimizes errors caused by rundown. However, the Kd calculated from the mean currents was similar. The mean SAC current was 2.04 ± 0.14 pA (SEM) over 11 pressure steps before GsMTx-4 application (Fig. 6 A), and fell to 0.17 ± 0.02 pA during toxin perfusion. (The average current over the last eight pressure steps, 10 s after GsMTx-4 washout, returned to the initial current level of 2.28 ± 0.17 pA.) For a single binding site, Michaelis-Menten kinetics predicts the ratio of the blocked to the unblocked current is I/I0 = 1(1 + K d/S), where S is the substrate (toxin) concentration and Kd is the equilibrium dissociation constant. Using the data from Fig. 6, I/I0 = 0.083, which gives a binding constant Kd = 415 nM, consistent with the value calculated from the ratio of association and dissociation rates.
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; Norton and Pallaghy 1998). GsMTx-4 shows <50% homology to any other peptide toxin. Other tarantula toxins that block voltage-gated Ca2+ and K+ channels show the highest percentage of similarity to GsMTx-4, as illustrated by the amino acid alignment in Fig. 8. A K+ channel toxin labeled protein 5 from Brachypelma smithii ( Kaiser et al. 1994; Norton and Pallaghy 1998) shows 50% total sequence similarity. The most significant regions of homology occur within the cysteine motif. Besides the conserved cysteine motif, there are three other residues (F4, D13, and L20) that are conserved in all five toxins. Like the positively charged 
-conotoxin and -agatoxin families of Ca2+ channel blockers, GsMTx-4 carries an overall positive charge (+5).
GsMTx-4 Effects on Astrocyte Whole-Cell Swelling-activated Currents
A large conductance increase occurs upon hypotonic swelling of neonatal astrocytes. Part of this current may be due to nonselective cationic SACs (Kimelberg and Kettenmann 1990). However, a large rapidly developing DIDS-sensitive anionic current dominates the passive membrane current (Pasantes-Morales et al. 1994; Bakhramov et al. 1995). After 30-s exposure to hypotonic conditions, adult astrocytes display a similar large-conductance increase that slowly inactivated at large depolarizing voltages [compare Fig. 9 A (resting current) to B (swelling-activated current)]. During hypotonic exposure, cells were held at –50 mV before I-V test voltage steps to reduce the influence of voltage-gated Ca2+ channels on Ca2+ influx. The swelling-activated current has a large anionic component since 50 µM DIDS produced a significant reduction in current (especially at depolarized potentials) and a –33-mV shift in reversal potential ( Fig. 10, n = 6). A residual current with a reversal potential shifted toward EK remained. Applying 5 µM GsMTx-4 while hypotonically swelling the cell significantly reduced the peak current response at 30 s after hypotonic exposure (Fig. 9 C). After washout of GsMTx-4, a hypotonic stimulus produced larger swelling-activated currents, although less than the original control stimulus (Fig. 9 D). This reduced response after washout is not due to lingering toxin effects, since >3 min of washout separated successive hypotonic stimuli. The response to successive hypotonic exposures slowly decreased over time (Fig. 9 F, a and b), probably due to RVD mechanisms. Representative peak-current responses from two different cells displayed a roughly linear decrease in swelling-activated current (Fig. 9 F, 
). GsMTx-4 always reduced the swelling-activated current from the control response (Fig. 9 F, 
). In light of the slowly degrading hypotonic response, to estimate the amount of GsMTx-4 block, we had to correct for the "rundown" by linear interpolation. The I-V profiles for the swelling-activated difference currents (Fig. 9 G) show a clear difference between the before () and after (•) responses. The percent block produced by GsMTx-4 () relative to each of the control curves is shown to the right. The estimated reduction in swelling-activated current produced by 5 µM GsMTx-4 was similar at both hyperpolarizing and depolarizing potentials (48% at –100 mV and 38% at +100 mV). Furthermore, unlike DIDS, which produced a large (–33 mV) shift in reversal potential due to the specific loss of anionic current, GsMTx-4 produces almost no change in reversal potential (+2 mV, statistically indistinguishable from 0 mV).
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; Clemo et al. 1998). Both an inwardly rectifying cation selective current (ICir,swell) and an outwardly rectifying Cl – current (ICl,swell) are involved in volume regulation in cardiac myocytes. In ventricular myocytes from rabbits with aortic regurgitation-induced congestive heart failure, these two currents are constitutively activated in isotonic media (Fig. 11 A, a) and are inactivated when the cell is perfused with hypertonic saline (b). At 0.4 µM, GsMTx-4 produced a nearly complete block of the inward ICir,swell, but had no effect on the outward ICl,swell (Fig. 11 B, c). However, whole-cell current was unaffected by GsMTx-4 when swelling-activated currents were inactivated by 1.5T hypertonic saline ( Fig. 11 B, d). The difference currents in Fig. 11 C show that GsMTx-4 blocked only inward swelling-activated current [compare Fig. 11 C, 1.0TC – 1.5TC (total I Cir,swell) with 1.0TC – 1.0TTx (toxin sensitive ICir,swell)]. The remaining inward current is largely ICl,swell. The toxin produced no further current reduction in the presence of hypertonic saline, which inactivates the swelling-activated current (Fig. 11 C, 1.5T C – 1.5TTx). This result again supports the specificity of GsMTx-4. When volume changes are examined (Fig. 11 D), GsMTx-4 produced a cell volume reduction that is 40% of that produced by 1.5T hypotonic saline.
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DISCUSSION |
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) suggests a clinical significance. Its ability to suppress cell swelling may also be clinically useful for dealing with edema. GsMtx-4 may be derivatized to make affinity columns for purifying the channel protein and for making histochemical markers to localize the channels. It is surprising that GsMtx-4 can act across different tissues in similar concentrations. This implies a strong homology between the cationic SACs of these tissues and may help to define a family of channels. The teleological significance of why a rather mild tarantula venom would have the ability to block SACs in a rabbit heart or a rat brain is unclear. Perhaps insects, the spider's normal prey, have similar channels.
The mechanism of action of GsMtx-4 remains to be determined. We do know that it can act on closed channels, but we don't know if this occurs because the activation curve is shifted to higher tensions or because GsMtx-4 blocks the permeation path. These studies are now in progress.
GsMTx-4 Structure
GsMTx-4 possesses an ICK consensus cysteine motif with the basic structure defined by three cysteine pairs (C1–C4 , C2–C5, and C3–C6 ) that stabilize a core region composed of a triple-stranded antiparallel β sheet (for review, see Norton and Pallaghy 1998). Enzymatic digest fragments are currently being analyzed to confirm this structure. Examples of toxin families active against ion channels that possess ICK motifs are: the µ-agatoxins and 
-atracotoxins, which block voltage-activated Na + channels; the -agatoxins, -conotoxins, and -atracotoxins, which block voltage-gated Ca2+ channels; and hanatoxin, 
-conotoxin, and TXP-5, which block voltage-gated K+ channels. GsMTx-4 shows the most sequence similarity to K+ and Ca2+ channel blocking toxins from tarantula venoms, the highest being TXP-5 from the Brachypelma smithii tarantula, where the similarity is 54%. However, sequence homology between ICK containing peptide toxins is a poor indicator of functional similarities. For example, the N-type Ca2+ channel blocker -conotoxin MVIIA has >80% sequence similarity with P/Q-type Ca2+ channel blocker -conotoxin MVIIC, while sharing only 45% sequence similarity with the N-type Ca2+ channel blocker -conotoxin GVIA.
We have recently produced a recombinant GsMTx-4 peptide in bacteria that in initial experiments blocks SACs in outside-out patches from astrocytes. This removes the possibility of a copurified contaminant along with GsMtx-4 from raw venom.
GsMTx-4 Binding Affinity in Astrocyte and Cardiac Myocyte Assays
The equilibrium constant for toxin binding was calculated to be 600 nM. While many peptide toxins are highly specific for their receptor, having affinities (IC50 or Kd) in the 0.1–100 nM range, GsMTx-4 binds 5–50x more tightly than any other antagonist tested to date on any stretch channel. It appears to be specific for cation SACs since it did not effect voltage-sensitive currents in astrocytes or ICl,swell in myocytes. The complete block of ICir,swell at 0.4 µM indicates that toxin affinity for its binding site in cardiac myocytes may be even stronger than in astrocytes. Preliminary results from atrial-induced fibrilation experiments with Langendorff-perfused rabbit hearts suggest that GsMTx-4 doesn't block normal electrical activity of the heart (eliminating many possible sites of cross reactivity) and the toxin may have a higher affinity for these cells in situ (Bode et al. 1999). In these studies, 0.17 µM GsMtx-4 was capable of inhibiting the atrial fibrillation associated with dilatation.
Changes in SAC Properties between Cell-attached and Outside-Out Patches
The properties of SACs in activated adult astrocytes, including ion selectivity, conductance, inward rectification, and adaptation, are similar to cation-selective SACs observed in other systems ( Yang and Sachs 1993). Outside-out patches were used in only one previous study to determine the effects of Gd3+ ( Yang and Sachs 1989) on SACs in Xenopus oocytes. However, in that report, the effects of outside-out formation on channel properties was not rigorously assessed because it was difficult to maintain activity in the patch. Patches from most cell types rapidly lose SAC activity with excision.
In the adult astrocytes, SAC adaptation is lost during outside-out patch formation, or if >10 mmHg suction is used for cell-attached seal formation. At voltages where adaptation should rapidly reduce channel Po (–50 mV), in outside-out patches we observed a delayed activation instead (Fig. 1 B, +100 mV average current, and 5 A, average current). This selective loss of adaptation is similar to the two stages of decoupling described by Hamill and McBride 1992 in Xenopus oocytes. The mechanisms for these changes in gating as a function of patch history remain to be determined.
However, the intrinsic permeation properties of the channels, such as channel conductance, rectification, and ion selectivity, seem less likely to be affected by cytoskeletal attachments and appear less sensitive to patch history, as shown in Fig. 1C and Fig. D. Even while more pressure/suction is required over time to activate the channel in either configuration (decoupling of the tonic gating element), channel conductance and rectification remain unchanged. Furthermore, although ion selectivity was not rigorously compared between the two patch configurations, channel conductance was 46 pS with 130 mM CsCl substituted for KCl in the pipette, demonstrating the channel is nonselective for cations in the cell-attached mode. Thus, outside-out patches are an adequate representation of the activity in cell-attached patches and a much more flexible preparation for screening.
SAC Activity during Astrocyte Cell Swelling
The sensory processes for RVD have not been determined, but these experiments strongly suggest that cationic SACs play a role. While dilution of internal K+ and an increase Na+ flux could contribute to membrane depolarization, this is an ineffective stimulus under voltage clamp and it has been demonstrated in multiple studies on different cell types that an increase in anionic current is the major contributor to membrane depolarization during hypotonic swelling ( Pasantes-Morales et al. 1994; Bakhramov et al. 1995). The trigger for the volume-activated anion current is still unclear. In our adult astrocytes, anion current accounts for nearly the entire reversal potential shift that occurs after hypotonic exposure. GsMTx-4 produces a 40–50% reduction in swelling-activated anion current, suggesting that cationic SACs contribute to activation of the current. However, in contrast to DIDS blockade of the anionic current, the reduction in swelling-activated current produced by GsMTx-4 showed no change in reversal potential. This could be due to a reduction of SAC cation current or an overall decrease in all swelling-activated currents. Dose response studies with GsMtx-4 are in order.
Although anion current dominates the whole-cell conductance during RVD, swelling-activated K+ currents that generally develop more slowly are rate limiting for Cl– efflux. Increasing the cation flux with gramicidin can circumvent the rate-limiting effect of the slowly increasing K+ current ( Pasantes-Morales et al. 1994). Activation of cation-selective SACs, like gramicidin, would increase the flux of cations (increasing Cl – efflux), and thus the rate of RVD.
RVD in astrocytes has been reported to be a Ca2+-dependent process (O'Connor and Kimelberg 1993; Bender et al. 1994). Thus, GsMTx-4 may block Ca2+ influx through SACs' reducing Ca2+-sensitive swelling-activated currents. Contributions to Ca2+ influx through voltage-gated Ca2+ channels were reduced by clamping the cell at –50 mV before recording the whole-cell currents. A number of swelling-activated anion currents have been identified that are induced by secondary messenger systems such as Ca2+, calmodulin, and various kinases. Although it has been shown that the DIDS-sensitive Cl– current is not Ca2+ dependent in neonatal astrocytes ( Pasantes-Morales et al. 1994; Bakhramov et al. 1995; Crepel et al. 1998), Ca2+-activated Cl– currents are partially responsible for the anion efflux during RVD in Ehrlich ascite cells ( Hoffmann et al. 1986; Lambert et al. 1989). It is possible that anion currents between neonatal and adult rat astrocytes may differ, especially since different SACs are observed between separate neonatal preparations ( Bowman et al. 1992; Islas et al. 1993) and adult astrocytes. Further characterization of GsMTx-4's effects on membrane currents and Ca2+ influx is required to more clearly define the role of SACs in RVD.
GsMTx-4 Effect on Rabbit CHF-model Cardiac Myocytes
In cardiac myocytes, stretch/swell-induced currents may play a critical role in the development of dysrhythmias and hypertrophy, and may alter contractile function. Cationic (ICir,swell) and anionic (I Cl,swell) swelling-activated currents have been identified in hypotonically swollen rabbit cardiac myocytes ( Clemo and Baumgarten 1997), and these may also be activated by the cell swelling that occurs during ischemia. Cardiac myocytes isolated from dog hearts with Tachychardia-induced CHF ( Clemo et al. 1998) maintain a cell volume 1.24x greater than normal cells. Both ICir,swell and ICl,swell are constitutively active in these CHF-model cardiomyocytes under isotonic conditions. Moreover, swelling-activated currents are persistently active in rabbit myocytes from an aortic regurgitation model. GsMTx-4 at 0.4 µM specifically blocked ICir,swell in CHF cardiomyocytes. The cation current ICir,swell might represent activity of cationic SACs. They are both inwardly rectifying, blocked by Gd3+ , and poorly selective for cations (PK/P Na = 6).
The fact that GsMTx-4 blocks SACs in rat astrocytes and ICir,swell (properties similar to cation-selective SACs) in rabbit cardiac myocytes suggests that many cell types incorporate SACs as part of the volume-regulatory process. Furthermore, the common toxin sensitivity suggests that at least some cation channels opened by direct mechanical stimulation are also opened by cell swelling. GsMTx-4 will be useful in elucidating the function of SACs in a variety of systems under physiologically normal and stressed conditions.
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ACKNOWLEDGMENTS |
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Submitted: 9 July 1999
Revised: 2 March 2000
Accepted: 6 March 2000
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TopABSTRACTINTRODUCTION METHODS RESULTS DISCUSSION REFERENCES |
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