Correspondence to: Thomas M. Suchyna, Dept. of Physiology and Biophysics, 320 Cary Hall, SUNY at Buffalo, Buffalo, NY 14214. Fax:716-829-2028 E-mail:suchyna{at}acsu.buffalo.edu.
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Abstract |
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We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spatulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying fractions on stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. Although the channel gating kinetics were different between cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance (~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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High-affinity inhibitory peptide toxins have proven to be powerful tools for elucidating the ion channel components of whole-cell currents. Stretch-activated channels (SACs)1 are the only major class of ion channels for which a specific inhibitor does not exist. Gd3+ is the best known blocker of SACs (Kds ranging from 1 to 100 µM) and is widely used to identify these channels. However, Gd3+ also blocks a variety of other channels such as L- and T-type Ca2+ (
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 (
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
A number of studies have suggested that nonselective cation-permeable SACs play a role in both membrane depolarization (
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
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 (
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METHODS |
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Toxin Isolation
Grammostola spatulata (Theraphosidae) spiders were obtained from a captive population at Hogel Zoo (Salt Lake City, UT). The Grammostola species have recently been reassigned to the genus Phixotricus (
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Further purification of pool 9 (see Fig 4 B) was achieved by RP chromatography on a Vydac C18 column (10 x 250 mm, 5 µm, 300 Å; The Separations Group) equilibrated in 10% solution B. Lyophilized pool 9 was dissolved in 4 ml of 10% solution B and chromatographed in 1-ml portions eluting with a 10-min gradient (1028% solution B), followed by a 64-min gradient (2860% solution B). The first gradient was begun 5 min after injection of the sample, the effluent was monitored at 220 nm, and three fractions were collected. Corresponding fractions from the four chromatographies were combined, lyophilized, and assayed as described above. Only pool B showed block of the SACs.
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Attempts to affect further purification of pool B by anion and cation exchange chromatography (MonoQ and MonoS resins; Pharmacia LKB Laboratories, Inc.) were unsuccessful. The material was not retained on the anion exchange column and retained too strongly on the cation exchange columns. The peptide did not elute with salt gradient from any of the cation exchange resins tried (02 M NaCl in 50 mM sodium phosphate buffer, pH 7.0). A broad peak of material did elute at ~pH 11 from the MonoS column with a gradient from pH 712 (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 (2027% solution B), followed by a 46-min gradient (2750% 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 (3247% 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 ionizationtime of flight) instrument operated in linear delayed extraction mode (50100 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 (
Astrocyte Cell Culture
Activated adult astrocytes, isolated from gelatin-sponge implants from adult Sprague-Dawley rat brains (
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 1090 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 ( (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 35 ms into voltage steps.
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Cardiac Myocyte Preparation
Ventricular myocytes were freshly isolated from New Zealand white rabbits with aortic regurgitation-induced congestive heart failure (
Cardiac Myocyte Electrophysiology and Volume Determination
For a detailed explanation of methods, see 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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SAC Properties in Activated Adult Rat Astrocytes: Cell-attached Versus Outside-Out
The most efficient method for screening multiple venom samples is to use outside-out patches. However, first it was necessary to show that SAC function is maintained in the outside-out patch configuration. To date, all studies investigating the properties and function of SACs in astrocytes have focused on neonatal preparations and C6 glioma cell lines (
The open probability (Po) was time and voltage dependent, displaying a fast adaptation (within 100 ms at hyperpolarized potentials) similar to that reported for Xenopus oocytes (
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
The single channel conductance and inward rectification observed here were similar to the properties reported for other members of the family of nonselective cation SACs (for review, see ) 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 (
Astrocyte SAC Pharmacology
To characterize the SACs of adult astrocyte, we examined a number of compounds purported to be active against SACs in other systems ( Table 1). Gd3+, the most commonly used SAC reagent, completely blocked the channels in adult astrocytes at 50 µM. Amiloride, which blocks epithelial Na+ channels with nanomolar affinity, and the endogenous SACs in oocytes and audiovestibular hair cells (
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Although NMDA-type glutamate receptors have not been observed in astrocytes, these channels do show stretch sensitivity in macropatches from neonatal neurons (
Some reports indicate that L-type Ca2+ channels are stretch sensitive (
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.
Determining the specificity of a pharmacologic agent is an unending project, and defined only for the systems tested. We tested the pseudosteady state I-V relationship as it related to voltage-sensitive channels. Using the perforated patch technique, 5 µM GsMTx-4 did not significantly change the I-V profile (Fig 7 A), suggesting that it did not interact with slow, voltage-dependent, channels. By comparison, 5 mM CsCl, which was shown not to effect SACs in patches, produced a significant decrease in current at hyperpolarized potentials (Fig 7 B), where a Cs+-sensitive inward-rectifying K channel is known to be activated (
Sequence and Disulfide Structure of GsMTx-4
MALDI-MS analysis showed the molecular weight of the native toxin was 4,093.90 (MH+ ion). The alkylated and reduced toxin displayed a peak at mass/charge 4,690, indicating three disulfide bonds or six cysteine residues were present. NH2-terminal sequencing was followed by sequencing of two different COOH-terminal fragments produced by enzymatic digests with BNPS-skatole and Asp-N. The peptide sequence produced had a predicted mass 4,022.86, which is 71.04 D less than that measured for the intact toxin. This difference supports the presence of a COOH-terminal alanine (71.09 D), even though alanine was clearly absent in the last cycle of the Asp-N digestion product. The mass accuracy of the MALDI-MS analysis is approximately ±0.5 D with internal calibration. The final sequence shown in Fig 8 is 35 amino acids in length with the C-term alanine added.
The six cysteine residue included in boxes form an ICK motif (CX3-7 CX3-6CX0-5CX1-4CX4-13C) commonly observed in many other peptide toxins from both terrestrial and aquatic animal venoms (-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 (). 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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GsMTx-4 Effects on CHF Model Ventricular Myocytes Whole-Cell Currents
Swelling-activated currents in rabbit and dog cardiac myocytes are persistently activated in CHF and may play a role in the development of congestive heart failure (
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DISCUSSION |
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A New Tool
We have found the first specific reagent for mechanosensitive ion channels (MSCs). It is surely only a prototype, with many reagents to follow. While studies are needed to establish the cross reactivity of GsMtx-4 for different types of MSCs, as well as for cross reactivity with different types of channels, GsMtx-4 is a unique agent. It can be used to test the involvement of SACs in physiological processes in situ or in vivo. Its ability to suppress cardiac arrhythmias (
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 (C1C4 , C2C5, and C3C6 ) that stabilize a core region composed of a triple-stranded antiparallel ß sheet (for review, see -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 0.1100 nM range, GsMTx-4 binds 550x 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 (
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 (
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
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 (
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 (
RVD in astrocytes has been reported to be a Ca2+-dependent process (
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 (
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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Footnotes |
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1 Abbreviations used in this paper: CHF, congestive heart failure; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid; ICK, inhibitor cysteine knot; RP, reverse phase; RVD, regulated volume decrease; SAC, stretch-activated channel; TFA, trifluoroacetic acid.
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Acknowledgements |
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This work was funded by grants to Dr. Frederick Sachs from the National Institutes of Health, the United States Army Research Office, and NPS Pharmaceuticals, Inc. Studies on myocytes were supported by HL46764 to Dr. Baumgarten.
Submitted: 9 July 1999
Revised: 2 March 2000
Accepted: 6 March 2000
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References |
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Bascur, L. , Yevenes, I. , Barja, P. 1982 . Effects of Loxosceles laeta spider venom on blood coagulation. Toxicon. 20:795-796 [Medline].
Ben-Tabou, S. , Keller, E. , Nussinovitch, I. 1994 . Mechanosensitivity of voltage-gated calcium currents in rat anterior pituitary cells. J. Physiol. 476:29-39 [Abstract].
Bender, A.S. , Mantelle, L.L. , Norenberg, M.D. 1994 . Stimulation of calcium uptake in cultured astrocytes by hypoosmotic stress: effect of cyclic AMP. Brain Res. 645:27-35 [Medline].
Bender, A.S. , Schousboe, A. , Reichelt, W. , Norenberg, M.D. 1998 . Ionic mechanisms in glutamate-induced astrocyte swelling: role of K+ influx. J. Neurosci. Res 52:307-321 [Medline].
Biagi, B.A. , Enyeart, J.J. 1990 . Gadolinium blocks low- and high-threshold calcium currents in pituitary cells. Am. J. Physiol. 259:C515-C520
Bode, F. , Sachs, F. , Franz, M.R. 1999 . Atrial fibrillation is inhibited by a peptide from spider venom that blocks stretch activated ion channels. Circulation 100:1784. (Abstr.)
Bowman, C.L. , Ding, J.P. , Sachs, F. , Sokabe, M. 1992 . Mechanotransducing ion channels in astrocytes. Brain Res. 584:272-286 [Medline].
Bowman, C.L. , Lohr, J.W. 1996 . Mechanotransducing ion channels in C6 glioma cells. Glia. 18:161-176 [Medline].
Chamberlin, M.E. , Strange, K. 1989 . Anisosmotic cell volume regulation: a comparative view. Am. J. Physiol. 257:C159-C173
Chen, Y. , Simasko, S.M. , Niggel, J. , Sigurdson, W.J. , Sachs, F. 1996 . Ca2+ uptake in GH3 cells during hypotonic swelling : the sensory role of stretch-activated ion channels. Am. J. Physiol. 270:C1790-C1798
Christensen, O. 1987 . Mediation of cell volume regulation by Ca2+ influx through stretch-activated channels. Nature. 330:66-68 [Medline].
Clemo, H.F. , Baumgarten, C.M. 1997 . Swelling-activated Gd3+-sensitive cation current and cell volume regulation in rabbit ventricular myocytes. J. Gen. Physiol. 110:297-312
Clemo, H.F. , Stambler, B.S. , Baumgarten, C.M. 1998 . Persistent activation of a swelling-activated cation current in ventricular myocytes from dogs with tachycardia-induced congestive heart failure. Circ. Res. 83:147-157
Crepel, V. , Panenka, W. , Kelly, M.E. , MacVicar, B.A. 1998 . Mitogen-activated protein and tyrosine kinases in the activation of astrocyte volume-activated chloride current. J. Neurosci. 18:1196-1206
Elinder, F. , Arhem, P. 1994 . The modulatory site for the action of gadolinium on surface charges and channel gating. Biophys. J. 67:71-83 [Abstract].
Fontana, A. 1972 . Modification of tryptophan with BNPS-Skatole (2-2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine). In Colowick S.P. , Kaplan N.O. , eds. Methods in Enzymology. Vol. 25, part B. New York, NY , Academic Press, Inc., 419-423 .
Hamill, O.P. , McBride, D.W. 1996 . The pharmacology of mechanogated membrane ion channels. Pharmacol. Rev. 48:231-252 [Abstract].
Hamill, O.P. , McBride, D.W., Jr. 1992 . Rapid adaptation of single mechanosensitive channels in Xenopus oocytes. Proc. Natl. Acad. Sci. USA. 89:7462-7466 [Abstract].
Hoffmann, E.K. , Lambert, I.H. , Simonsen, L.O. 1986 . Separate, Ca2+-activated K+ and Cl - transport pathways in Ehrlich ascites tumor cells . J. Membr. Biol. 91:227-244 [Medline].
Hoffmann, E.K. , Simonsen, L.O. 1989 . Membrane mechanisms in volume and pH regulation in vertebrate cells . Physiol. Rev. 69:315-381
Horn, R. , Marty, A. 1988 . Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen. Physiol. 92:145-159 [Abstract].
Hu, H. , Sachs, F. 1997 . Stretch-activated ion channels in the heart. J. Mol. Cell. Cardiol. 29:1511-1523 [Medline].
Islas, L. , Pasantes-Morales, H. , Sanchez, J.A. 1993 . Characterization of stretch-activated ion channels in cultured astrocytes. Glia. 8:87-96 [Medline].
Jorgensen, F. , Ohmori, H. 1988 . Amiloride blocks the mechano-electrical transduction channel of hair cells of the chick. J. Physiol. 403:577-588 [Abstract].
Kaiser, I.I. , Griffin, P.R. , Aird, S.D. , Hudiburg, S. , Shabanowitz, J. , Francis, B. , John, T.R. , Hunt, D.F. , Odell, G.V. 1994 . Primary structures of two proteins from the venom of the Mexican red knee tarantula (Brachypelma smithii). Toxicon. 32:1083-1093 [Medline].
Kimelberg, H.K. 1995 . Current concepts of brain edema. Review of laboratory investigations. J. Neurosurg. 83:1051-1059 [Medline].
Kimelberg, H.K. , Anderson, E. , Kettenmann, H. 1990 . Swelling-induced changes in electrophysiological properties of cultured astrocytes and oligodendrocytes. II. Whole-cell currents. Brain Res. 529:262-268 [Medline].
Kimelberg, H.K. , Kettenmann, H. 1990 . Swelling-induced changes in electrophysiological properties of cultured astrocytes and oligodendrocytes. I. Effects on membrane potentials, input impedance and cellcell coupling. Brain Res. 529:255-261 [Medline].
Kluesener, B.G. , Boheim, H. , Liss, J. , Engelberth,, Weiler, E.W. 1995 . Gadolinium-sensitive, voltage-dependent calcium release channels in the endoplasmic reticulum of a higher plant mechanoreceptor organ. EMBO (Eur. Mol. Biol. Organ.) J. 14:2708-2714 [Abstract].
Lambert, I.H. , Hoffmann, E.K. , Jorgensen, F. 1989 . Membrane potential, anion and cation conductances in Ehrlich ascites tumor cells. J. Membr. Biol. 111:113-131 [Medline].
Lampe, R.A. , Defeo, P.A. , Davison, M.D. , Young, J. , Herman, J.L. , Spreen, R.C. , Horn, M.B. , Mangano, T.J. , Keith, R.A. 1993 . Isolation and pharmacological characterization of omega-grammotoxin SIA, a novel peptide inhibitor of neuronal voltage-sensitive calcium channel responses. Mol. Pharmacol. 44:451-460 [Abstract].
Lane, J.W. , McBride, D. , Hamill, O.P. 1991 . Amiloride block of the mechanosensitive cation channel in Xenopus oocytes. J. Physiol. 441:347-366 [Abstract].
Langan, T.J. , Plunkett, R.J. , Asada, H. , Kelly, K. , Kaseloo, P. 1995 . Long-term production of neurotrophic factors by astrocyte cultures from hemiparkinsonian rat brain. Glia. 14:174-184 [Medline].
Langton, P.D. 1993 . Calcium channel currents recorded from isolated myocytes of rat basilar artery are stretch sensitive. J. Physiol. 471:1-11 [Abstract].
Magid, N.M. , Young, M.S. , Wallerson, D.C. , Goldweit, R.S. , Carter, J.N. , Devereux, R.B. , Borer, J.S. 1988 . Hypertrophic and functional response to experimental chronic aortic regurgitation. J. Mol. Cell Cardiol. 20:239-246 [Medline].
Narasimhan, L. , Singh, J. , Humblet, C. , Guruprasad, K. , Blundell, T. 1994 . Snail and spider toxins share a similar tertiary structure and "cystine motif". Nat. Struct. Biol. 1:850-852 [Medline].
Newcomb, R. , Szoke, B. , Palma, A. , Wang, G. , Chen, X. , Hopkins, W. , Cong, R. , Miller, J. , Urge, L. , Tarczy-Hornoch, K. et al. 1998 . Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas. Biochemistry. 37:15353-15362 [Medline].
Norton, R.S. , Pallaghy, P.K. 1998 . The cystine knot structure of ion channel toxins and related polypeptides. Toxicon. 36:1573-1583 [Medline].
O'Connor, E.R. , Kimelberg, H.K. 1993 . Role of calcium in astrocyte volume regulation and in the release of ions and amino acids. J. Neurosci. 13:2638-2650 [Abstract].
Paoletti, P. , Ascher, P. 1994 . Mechanosensitivity of NMDA receptors in cultured mouse central neurons. Neuron. 13:645-655 [Medline].
Pasantes-Morales, H. 1996 . Volume regulation in brain cells: cellular and molecular mechanisms. Metab. Brain Dis. 11:187-204 [Medline].
Pasantes-Morales, H. , Murray, R.A. , Lilja, L. , Moran, J. 1994 . Regulatory volume decrease in cultured astrocytes. I. Potassium- and chloride-activated permeability. Am. J. Physiol. Cell Physiol. 266:C165-C171
Perez, F. , Luca, S. , DaSilva, P. , Bertani, R. 1996 . Systematic revision and cladistic analysis of Theraphosinae. Mygalomorph. 1:33-68 .
Ruknudin, A. , Sachs, F. , Bustamante, J.O. 1993 . Stretch-activated ion channels in tissue-cultured chick heart. Am. J. Physiol. Circ. Physiol. 264:H960-H972 .
Sachs, F. 1988 . Mechanical transduction in biological systems. Crit. Rev. Biomed. Eng. 16:141-169 [Medline].
Sachs, F. 1992 . Stretch sensitive ion channels: an update. In Corey D.P. , Roper S.D. , eds. Sensory Transduction. New York, NY , Rockefeller University Press, 241-260 .
Sachs, F. , Morris, C. 1998 . Mechanosensitive ion channels in non specialized cells. In Blaustein M.P. , Greger R. , Grunicke H. , Jahn R. , Mendell L.M. , Miyajima A. , Pette D. , Schultz G. , Schweiger M. , eds. Reviews of Physiology and Biochemistry and Pharmacology. Berlin, Germany , Springer Verlag, 1-78 .
Sarkadi, B. , Parker, J.C. 1991 . Activation of ion transport pathways by changes in cell volume . Biochim. Biophys. Acta. 1071:407-427 [Medline].
Silberberg, S.D. , Magleby, K.L. 1997 . Voltage-induced slow activation and deactivation of mechanosensitive channels in Xenopus oocytes. J. Physiol. 505:551-569 [Abstract].
Small, D.L. , Morris, C.E. 1995 . Pharmacology of stretch-activated K+ channels in Lymnaea neurones. Br. J. Pharmacol. 114:180-186 [Abstract].
Sontheimer, H. 1992 . Astrocytes, as well as neurons, express a diversity of ion channels . Can. J. Physiol. Pharmacol. 70:S223-S238 [Medline].
Swartz, K.J. , MacKinnon, R. 1995 . An inhibitor of the Kv2.1 potassium channel isolated from the venom of a Chilean tarantula. Neuron. 15:941-949 [Medline].
Tazaki, M. , Suzuki, T. 1998 . Calcium inflow of hamster Merkel cells in response to hyposmotic stimulation indicate a stretch-activated ion channel. Neurosci. Lett. 243:69-72 [Medline].
Vandenberg, J.I. , Rees, S.A. , Wright, A.R. , Powell, T. 1996 . Cell swelling and ion transport pathways in cardiac myocytes. Cardiovasc. Res. 32:85-97 [Medline].
Wilson, K.J. 1989. Methods in protein sequence analysis: proceedings of the 7th international conference, Berlin, July 38, 1988. B. Wittmann-Leibold, editor. Springer-Verlag, New York, NY. 310330.
Yang, X.C. , Sachs, F. 1989 . Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science. 243:1068-1071 [Medline].
Yang, X.C. , Sachs, F. 1993 . Mechanically sensitive, non-selective, cation channels. In Siemen D. , Hescheler J. , eds. Non-Selective Ion Channels. Heidelberg, Germany, Springer-Verlag, 79-92 .