Tityustoxin-K(alpha) blockade of the voltage-gated potassium channel Kv1.3

Abstract

1. We investigated the action of TsTX-Kalpha on cloned Kv1.3 channels of the Shaker subfamily of voltage-gated potassium channels, using the voltage-clamp technique. Highly purified TsTX-Kalpha was obtained from the venom of the Brazilian scorpion Tityus serrulatus using a new purification protocol. Our results show that TsTX-Kalpha blocks Kv1.3 with high affinity in two expression systems. 2. TsTX-Kalpha blockade of Kv1.3 channels expressed in Xenopus oocytes was found to be completely reversible and to exhibit a pH dependence. The K(D) was 3.9 nM at pH 7.5, 9.5 nM at pH 7.0 and 94.5 nM at pH 6.5. 3. The blocking properties of TsTX-Kalpha in a mammalian cell line (L929), stably transfected to express Kv1.3, were studied using the patch-clamp technique. In this preparation, the toxin had a K(D) of 19.8 nM at pH 7.4. 4. TsTX-Kalpha was found to affect neither the voltage-dependence of activation, nor the activation and deactivation time constants. The block appeared to be independent of the transmembrane voltage and the toxin did not interfere with the C-type inactivation process. 5. Taken as a whole, our findings indicate that TsTX-Kalpha acts as a simple blocker of Kv1.3 channels. It is concluded that this toxin is a useful tool for probing not only the physiological roles of Kv1.2, but also those mediated by Kv1.3 channels.

Figure 1

Reverse-phase HPLC of fraction XI (0.3 mg). Adsorbed proteins were eluted with a linear acetonitrile gradient (0–60%) in 0.1% (v/v) trifluoroacetic acid at a flow rate of 1.0 ml min⁻¹. Absorbance was monitored at 214 nm. Buffer B was 60% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid. The peak XI-4 corresponds to TsTX-Kα. Inset, PAGE on a 10% (w/v) acrylamide gel at pH 4.5. Lane 1: T. serrulatus whole venom; lane 3: fraction XI-2; lane 4: fraction XI-3; lanes 5, 10 and 11: fraction XI-4; lane 6: fraction XI-5; lane 7: fraction XI-6; lane 8: fraction XI.

Figure 2

Basic properties of TsTX-Kα blockade of currents through cloned mKv1.3 channels expressed in Xenopus oocytes. The currents were elicited by pulses ranging from −50 to +50 mV in 10 mV steps from a holding potential of −80 mV, in the absence (left) and presence (right) of the toxin in the bath solution, which had the pH adjusted to either 7.5 (a) or 6.5 (b). The interpulse interval was 30 s. Lowpass filtering was at 2 and 1 kHz and the sampling rate 10 and 5 kHz in (a) and (b), respectively. (c) Dose–response curves determined at different pH. The fraction of unblocked current (I_Toxin/I_Control) was calculated by measuring the peaks of currents elicited by voltage steps from −80 to +40 mV. Bars indicate s.e.m., and the number of repetitions averaged, if not exactly three, are given next to the data points. The solid curves represent fit of the equation: I_Toxin/I_Control=K_D/(K_D+[T_x]) to the experimental points, where [T_x] indicates the toxin concentration and K_D is the dissociation constant. The best fit predicts a K_D of 3.9, 9.5 and 94.5 nM for pH 7.5, 7.0 and 6.5, respectively. (d) Reversibility of the block of Kv1.3 channels by TsTX-Kα (pH 7.5). Peak currents elicited by pulses from −80 to +40 mV, applied every 20 s, were normalized to the maximum value and plotted against time in control and in the presence of toxin.

Figure 3

Effects of TsTX-Kα on Kv1.3 currents in L929 cells. (a) Whole-cell currents elicited by voltage pulses ranging from −60 to +60 mV, in 15 mV steps, from a holding potential of −80 mV, in the absence (left) and presence (middle) of the toxin. The interpulse-interval was 30 s. The resultant current–voltage relationships, taken from the peak currents, are shown on the right-hand side. (b) The plot on the left-hand side shows the conductance–voltage relation for the K⁺ currents presented in (a). Fit lines are from a Boltzmann function, as described in the text. Values for V₀ and K are −30.0 and 5.9 mV in the absence and −27.3 and 6.2 mV in the presence of TsTX-Kα, respectively. The center panel shows tail currents recorded from the same cell at voltages between −85 and −60 mV, in 5 mV steps, after applying a 10 ms pulse to +40 mV from a holding potential of −90 mV. On the right-hand side, the peak tail currents in both conditions are plotted against voltage. The reversal potential, determined by a linear fit, was −76.7±6.7 mV for control and −76.5±5.7 mV in the presence of toxin (n=3). Lowpass filtering was at 2 and 5 kHz and the sampling rate 10 and 20 kHz in (a) and (b), respectively.

Figure 4

Concentration-dependent inhibition of Kv1.3 currents in L929 cells (a). The fraction of unblocked current was calculated as I_Toxin/I_Control measured at the peak of depolarizing steps from −80 to +40 mV. Bars indicate s.e.m. (n=4). The solid curves represent the fits obtained as described in Figure 2 giving a K_D of 19.8 nM. (b) Block of Kv1.3 channels by TsTX-Kα is voltage-independent. The percentage of inhibition was calculated according to the formula (1−I_Toxin/I_Control) × 100, where the peak currents in the presence and absence of the toxin were measured by depolarization to the indicated voltages from a holding potential of −80 mV (n=5 L929 cells).

Figure 5

TsTX-Kα does not affect the opening (a) and closing (b) kinetics of Kv1.3-mediated currents in L929 cells. Activation and deactivation time constants were calculated by fitting single-exponential functions to the current records in the presence and absence of the toxin. (a) Cells were depolarized to voltages between −15 and +60 mV, from a holding potential of −90 mV, at intervals of 20 s. Fits were from 10 to 90% of the current amplitude, as shown in the record to −15 mV. The plot on the right-hand side shows mean±s.e.m. of the activation time constants under control conditions and in the presence of TsTX-Kα (n=5). (b) Tail currents were elicited by voltage steps ranging from −80 to −20 mV, after a short activating pulse (10 ms) to +40 mV. On the right, mean± s.e.m. of the decay time constants of the tail currents, with and without toxin, are presented (n=5). All records were filtered at 5 kHz and sampled at 20 kHz.

Figure 6

The inactivation time constants of Kv1.3 currents in L929 cells are not affected by TsTX-Kα. (a) Currents recorded with 5 s pulses to +40 mV from a holding potential of −80 mV in the absence and presence of 20 nM TsTX-Kα. The first 1600 ms are shown in order to best visualize the double exponential fits to the decay phase of the current. The fit begins at 15 ms and ends at 1500 ms after the start of the pulse. The inset shows normalized full traces (control and toxin) almost exactly superimposed. (b) Inactivation time constants (mean±s.e.m., n=7) obtained as in (a). τ₁ is the slow component, while τ₂ is the fast component.