Identification of a peptide toxin from Grammostola spatulata spider venom that blocks cation-selective stretch-activated channels

Abstract

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 ( approximately 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 approximately 630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces approximately 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.

Figure 1

Cell-attached and outside-out patches from adult activated astrocytes showing stretch-sensitive channels with similar unitary conductance profiles but different gating properties. Representative single-channel current recordings are shown above average patch currents from a cell-attached patch (A) containing a single channel, and an outside-out patch (B) containing two to three channels. Cell-attached patch recordings were made with 140 mM KCl pipette saline, and outside-out patch recordings are with symmetrical 140-mM KCl pipette solutions. Pressure steps (indicated by the bar at the top) were applied to the patches at different holding potentials shown to the left of each recording. Voltages are relative to the extracellular side. Average current records were calculated from multiple pressure steps (ranging from 5 to 15 steps) at each voltage. In cell-attached mode, channel adaptation, lower P _o, and multiple subconductance states are apparent at negative potentials. Channels in outside-out patches from astrocytes show slow voltage-dependent activation and lower P _o at negative potentials. Unitary current–voltage plots were fitted with a second-order polynomial and show inward rectification for channels in cell-attached (C, n = 11) and outside-out (D, n = 16) patches. Voltages for cell-attached data points were corrected for the average resting membrane potential measured in the whole cell configuration. Each point represents an average current calculated by applying multiple pressure steps to a single patch.

Figure 2

External ion substitution shows SACs are cation selective in outside-out patches. The pipette saline was 140 mM KCl. Each data point represents the average current from multiple pressure steps to a single patch. The data sets were fitted with second-order polynomials. Data for symmetrical 140 mM KCl (□, solid line fit) are the same as shown in Fig. 1 D. Switching from 140 mM KCl to 140 mM NaCl (▵, dotted line fit) in the bath reduced the conductance at −100 mV from 44 to 33 pS, while there was no change in conductance at +100 mV (n = 10 patches). This suggests a weak selectivity for K⁺ over Na⁺. When external Cl⁻ is replaced with a less permeable anion gluconate (♦, dashed line fit), there was a negligible reduction in conductance at −100 mV (41 pS), and no change in conductance at +100 mV (n = 4 patches). However, when external K⁺ is replaced with the impermeant cation NMDG (▾, dash-dot line fit), the conductance was reduced to 5 pS at −100 mV (n = 3 patches).

Figure 3

Reverse-phase HPLC chromatograms showing sequential purification steps for identification of GsMTx-4 peptide. The percent acetonitrile that corresponds to specific venom peaks is indicated by the dotted line shown overlaying each chromatogram. A chromatogram of Grammostola whole venom (A) produced by a 40-min linear gradient from 15 to 55% acetonitrile at a flow rate of 3.5 ml/min. 1–11, labeled at the bottom, designate fractions pooled for testing on outside-out patches. The lines within the chromatogram mark the boundaries of each fraction. Fraction 9 (A) contained SAC blocking activity and was further fractionated in B. (B) Only fraction B showed SAC blocking activity and was further purified in C. 10 μg of the final material used in all experiments was run on a 25-min 32–47% linear gradient of acetonitrile at a flow rate of 1 ml/min (D).

Figure 4

GsMTx-4 blocks SACs in outside-out patches. SAC activity in response to pressure steps applied to an outside-out patch before and during GsMTx-4 application, and after washout are shown. The patch was held at −50 mV, and the pressure pulse is shown above the records. The entire experiment is comprised of 60 pressure steps: steps 1–20 occur before GsMTx-4 application, 21–38 while GsMTx-4 is being perfused, and 39–60 occur during washout. Each 500-ms pressure step was separated by 1.5 s at 0 pressure. Four representative records from each stage of the experiment are displayed.

Figure 5

GsMTx-4 rate of blocking determined by superfusion of activated SACs in outside-out patches. Average SAC currents calculated from 3-s pressure steps are indicated by the bars above the traces (A). The control trace was generated from 37 pressure steps applied to seven different patches held at −50 mV, with pressure levels ranging from 35 to 70 mmHg. The current increased exponentially over the 3-s pressure application. The GsMTx-4 response was produced by applying 5 μM toxin 1 s after the onset to the pressure step indicated by the GsMTx-4 bar. The GsMTx-4 current record was averaged from 29 pressure steps to six different patches held at −50 mV, with the steps ranging between 38 and 80 mmHg. Currents were nearly identical over the first second of the average current records, as shown when the two are superimposed in B. Subtracting the control current trace from the GsMTx-4 trace produced the difference current in C. The current trace during GsMTx-4 application was fitted with a single exponential yielding a time constant of 594 ± 10 ms (D). The fit is shown displaced from the data for clarity.

Figure 6

The GsMTx-4 dissociation rate was determined from the recovery rate of SAC current on washout. SAC currents were activated by 500-ms pressure steps at 2-s intervals in outside-out patches held at −50 mV. (A) average current (▪ ± SEM) from seven different patches. Channel current drops to the noise level rapidly upon application of toxin, and shows a slow recovery to the initial current level upon toxin washout. The recovery kinetics were fitted to a single exponential with a time constant of 4.7 ± 1.7 s (B).

Figure 7

GsMTx-4 does not significantly affect voltage-sensitive currents. Whole-cell currents are shown from astrocytes voltage clamped using the perforated-patch technique. (A) Average whole-cell current from six cells, produced by a 600-ms ramp from −120 to 80 mV. There is no significant difference between whole-cell currents in isotonic saline (♦) and currents measured between 30 and 120 s after perfusion with 5 μM GsMTx-4 (⋄). (B) Peak currents from a single cell stepped in 20-mV increments between −120 and 120 mV in the absence (♦) or presence (⋄) of 5 mM CsCl. In contrast to GsMTx-4, CsCl produces a significant decrease in current at hyperpolarized potentials. After the first application of CsCl, the cell was washed and the experiment was repeated. Thus, the I-V plot shows two sets of data points for both control and CsCl.

Figure 8

Sequence of GsMTx-4 showing homology to other ion channel peptide toxins. Cysteine motif residues are included in boxes. Dark shaded residues in the comparison peptide sequences are identical to GsMTx-4, while lighter shaded residues are similar. TXP5, K⁺ channel blocker ( Kaiser et al. 1994): 40% identity, 54% similarity; SNX-482, blocks E-type Ca²⁺ channels ( Newcomb et al. 1998): 40% identity, 49% similarity; ω-GramTX S1A, blocks N-, P-, and Q-type Ca²⁺ channels (not L-type) (Lampe et al. 1993): 34% identical, 46% similarity; Hanatoxin, K⁺ channel blocker ( Swartz and MacKinnon 1995): 28% identical, 37% similarity.

Figure 9

GsMTx-4 reduces whole-cell swelling-activated current in astrocytes exposed to hypotonic saline. Whole cell currents (A–D) from perforated patches on astrocytes. (A) Resting whole-cell currents in isotonic saline produced by the waveform shown in E (isotonic saline is normal bath saline with 80 mM NaCl replaced by 160 mM mannitol). Current scale bar is shown (right). Swelling-activated currents were recorded after the cell had been exposed for 30 s to hypotonic saline (B, isotonic saline minus 140 mM mannitol). (C) Perfusion of hypotonic saline with 5 μM GsMTx-4 produced an ∼75% reduction in the peak swelling-activated current at 30 s, after subtracting resting current. Swelling currents partially recovered ∼4 min after washout of GsMTx-4 (D). Peak swelling-activated currents at 100 mV (F, ▪) from two different cells (a and b) decreased over successive exposures to hypotonic solution. (F, ⋄) Peak currents measured during hypotonic exposures with GsMTx-4 present were reduced from the control. (G) I-V plot of the average swelling-activated peak currents from six cells measured 30–40 s after hypotonic exposure. The data points represent difference currents calculated by subtracting the resting current from hypotonic current. Control hypotonic current (▪), hypotonic currents in the presence of GsMTx-4 (⋄), and hypotonic currents after ∼5 min of washout (•). The hypotonic current in the presence of GsMTx-4 is ∼38% lower than control swell currents at +100 mV and ∼48% lower at −100 mV.

Figure 10

DIDS reduces swelling-activated currents in adult astrocytes. Whole-cell currents produced by a 600-ms ramp in voltage from −120 to 80 mV at 30 s after exposure to hypotonic saline are shown. Hypotonic and hypotonic + 50 μM DIDS difference current are shown, produced by subtracting the whole-cell current under isotonic conditions from the current observed during hypotonic exposure. A large reduction in swelling-activated current is observed at hyperpolarized potentials compared with the reduction observed at negative potentials. The reversal potential shift caused by DIDS is approximately −30 mV. The average reversal potential shift from six cells exposed to DIDS during hypotonic swelling was approximately −33 ± 3.5 mV.

Figure 11

Ionic currents (A–C) and cell volumes (D) measured during perforated patch voltage clamp (E_hold = −80 mV) of ventricular myocytes from rabbits with aortic regurgitation-induced CHF. Myocytes were exposed to 1.0T and 1.5T solution in the absence [1.0T_C (dashed line), 1.5T_C (solid line)] and presence [1.0T _Tx (dashed line), 1.5T_Tx(solid line)] of 0.4 μM GsMTx-4. (A) Osmotic shrinkage in the control solution reduced both inward and outward currents. (B) Toxin reduced the inward currents in 1.0T, but the currents in 1.5T were unaffected (compare A and B). (C) Difference currents: 1.0T_C–1.5T_C (dash-dot line), shrinkage-sensitive current due to inhibition of cationic SACs and anionic swelling currents; 1.0T_C–1.0T_Tx (dashed line), inwardly rectifying toxin-sensitive current. The toxin-sensitive current was similar to the Gd³⁺-sensitive currents recorded in the same model (data not shown, Clemo and Baumgarten, unpublished observations). 1.5T_C–1.5T_Tx (solid line), toxin did not affect membrane currents when SACs were inhibited by osmotic shrinkage. (D) Consistent with block of ion influx via cationic SACs, toxin reduced cell volume by 7% in 1.0T solution. In contrast, cell volume was unaffected after osmotic shrinkage in 1.5T solution, conditions under which SACs are closed. I-V curves elicited by ramp clamps (28 mV/s) and cell volume data are averages from five cells. Lower case letters (a, b, c, and d) designate time of current measurements in I-V profiles.