Biophys J. 2004 Jan;86(1 Pt 1):191-209.

The binding of kappa-Conotoxin PVIIA and fast C-type inactivation of Shaker K+ channels are mutually exclusive.

Source

Max-Planck-Institute for Experimental Medicine, Molecular and Cellular Neuropharmacology Group, Göttingen, Germany.

Abstract

Kappa-conotoxin PVIIA (kappa-PVIIA), a 27-amino acid peptide identified from the venom of Conus purpurascens, inhibits the Shaker K+ channel by blocking its outer pore. The toxin appears as a gating modifier because its binding affinity decreases with relatively fast kinetics upon channel opening, but there is no indication that it interferes with the gating transitions of the wild-type channels (WT), including the structural changes of the outer pore that underlie its slow C-type inactivation. In this report we demonstrate that in two outer pore mutants of Shaker-IR (M448K and T449S), that have high toxin sensitivity and fast C-type inactivation, the latter process is instead antagonized by and incompatible with kappa-PVIIA binding. Inactivation is slowed by the necessary preliminary unbinding of kappa-PVIIA, whereas toxin rebinding must await recovery from inactivation causing a double-exponential relaxation of the second response to double-pulse stimulations. Compared with the lack of similar effects in WT, these results demonstrate the ability of peptide toxins like kappa-PVIIA to reveal possibly subtle differences in structural changes of the outer pore of K+ channels; however, they also warn against a naive use of fast inactivating mutants as models for C-type inactivation. Unfolded from the antagonistic effect of inactivation, toxin binding to mutant noninactivated channels shows state- and voltage-dependencies similar to WT: slow and high affinity for closed channels; relatively fast dissociation from open channels at rate increasing with voltage. This supports the idea that these properties depend mainly on interactions with pore-permeation processes that are not affected by the mutations. In mutant channels the state-dependence also greatly enhances the protection of toxin binding against steady-state inactivation at low depolarizations while still allowing large responses to depolarizing pulses that relieve toxin block. Although not obviously applicable to any known combination of natural channel and outer-pore blocker, our biophysical characterization of such highly efficient mechanism of protection from steady-state outer-pore inactivation may be of general interest.

PMID:: 14695262; [PubMed - indexed for MEDLINE]
PMCID:: PMC1303782

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FIGURE 7

Dose-dependence of the effect of κ-PVIIA on T449S currents. (A) Current responses elicited by step-depolarizations to +40 mV in toxin-free conditions (Control) and in presence of 1 μM or 3 μM κ-PVIIA in the external solution. Increasing toxin concentration, [T], leads to a progressive reduction in peak currents and slowing of the inactivation. The decay phase of the control record is fitted by a double-exponential function with slow and fast rates, λ_s = 12.2 s⁻¹ and λ_f = 111 s⁻¹; the fitted rates of the dominant slow component of the toxin records are 10.6 s⁻¹ for [T] = 1 μM and 8.1 s⁻¹ for [T] = 3 μM. (B) Estimates of the unblock probability of activated channels at 40 mV, U^(A), derived from the decrease of λ_s values (see text), are plotted as a function of [T]. The solid line is the best fit of the data with the function U^(A) = K^(A)/(K^(A) + [T]) with an apparent dissociation constant K^(A) = 3.95 μM and n = 14 at 0.3, 10 at 1, 6 at 3, and 3 at 10 M. (C) Comparison of the early phases of the current responses shown in A; the delayed onset of the currents in the presence of 1 and 3 μM κ-PVIIA is fitted by the convolution of control activation and toxin-binding relaxation according to Eq. 4 (see text). The fitted binding-relaxation rates are: 1 μM, λ_B = 610 s⁻¹ and 3 μM, λ_B = 820 s⁻¹. (D) [T]-dependence of estimated toxin-binding kinetics at +40 mV. (Unfilled circles) λ_B estimates from the delay of early currents. (Solid circles) The off rate estimated as the product of λ_B × the unblock probability, U^(A), derived from the slowing of inactivation as in A and B. (Unfilled squares) The on rates estimated as λ_B × (1 − U^(A)). The straight lines are best fits according to λ_B = k_off + k_on × [T], with off = k_off, on = k_on × [T], k_off × s = 600 ± 40 s⁻¹, and k_on = 140 ± 30 μM⁻¹ s⁻¹. Data is given as mean ± SD; n = 14 at 300 nM, 10 at 1 μM, and 6 at 3 μM.

The Binding of κ-Conotoxin PVIIA and Fast C-Type Inactivation of Shaker K+ Channels are Mutually Exclusive

Biophys J. Biophys J;86(1):191-209.

SCHEME 3

Terlau et al. (1999) showed that in Shaker channels the recovery from N-type inactivation and the rebinding of κ-PVIIA could be simply unfolded by assuming their independence, supporting the view that the toxin does not distinguish between different nonconducting states of the channels if the closed gate is on the intracellular side. Such assumption is clearly not tenable in the case of M448K channels: as shown by Fig. 3 E, the apparent rebinding estimated by normalizing the early P2 responses under toxin to those of control would show a fairly large and unaccountable delay. The double-exponential relaxation of Fig. 3 D can instead be accounted for by a simple kinetic scheme in which toxin binding is incompatible with inactivation and that comprises only three statistically significant states (see Scheme 3 in Appendix): an inactivated state, an unblocked state (activatable for conductance), and a toxin-liganded state (activatable, but unavailable for conductance). If we denote with λ_I the rate characterizing the single exponential recovery of activatable currents in toxin-free conditions, and with λ_R=off + on the rate of toxin binding relaxations, such scheme predicts that during repolarizations the fraction U(t) of channels available for conduction follows a double-exponential time course with decaying rates λ_I and λ_R, approaching asymptotically a resting value U_C = off/(off + on). Even more stringently, for initial conditions in which the channels are all inactivated, it predicts that all four parameters that characterize the expression of U(t) are determined by the sole off and on binding rates according to (see Appendix) (2)

with

Confirming the above predictions, the smooth line in Fig. 3 D was obtained as the best fit of the data with Eq. 2 fixing λ_I to the value of control and allowing only on and off as free parameters, estimated as off = 0.3 s⁻¹ and on = 0.42 s⁻¹.

The Binding of κ-Conotoxin PVIIA and Fast C-Type Inactivation of Shaker K+ Channels are Mutually Exclusive

Biophys J. Biophys J;86(1):191-209.

FIGURE 6

Concentration and voltage-dependence of κ-PVIIA binding relaxations to open M448K channels. (A) Effect of κ-PVIIA on M448K currents elicited by +40 mV pulses. Current traces resulting from pulses to +40 mV in the absence (Control) and presence of 300 nM, 1 μM, or 3 μM κ-PVIIA in the external solution. Increasing toxin concentration leads to a reduction in peak currents and to an increased slowing of the inactivation. The solid lines, almost indistinguishable from raw data, are either a monoexponential fit of the inactivation decay for t > 2 × t_peak (Control), or double-exponential fits of the biphasic time course of toxin records in the same time interval. The time constants for the late inactivation are: Control, 11.7 ms; 300 nM κ-PVIIA, 21.3 ms; 1 μM κ-PVIIA, 26.7 ms; and 3 μM κ-PVIIA, 40.3 ms. The dashed line corresponds to 0 currents. (B) M448K-mediated currents upon stimulation to 0, +20, +40, and +60 mV from a holding potential of −100 mV in the presence of 1 μM κ-PVIIA. Notice the increase in the rate of inactivation at higher test voltages. Data are from the same oocyte of Fig. 6 A. The dashed line corresponds to 0 currents. (C) Voltage-dependence of the rate-constants characterizing inactivation and κ-PVIIA binding to open M448K channels. (Unfilled triangles) Inactivation rate of control currents, λ_h × s. (Unfilled squares) Second-order association rate-constant, k_on × μM × s. (Solid squares) First-order dissociation rate-constant, k_off × s. The k_on and k_off estimates were obtained by unfolding toxin-binding relaxations from inactivation as described in the text. λ_h and k_on data are connected by simple line segments. The thicker line through k_off data is a least-squares fit with the function k_off (V) = k_off(0) × exp(V/v_s). The k_on data do not show any systematic voltage-dependence and have a mean value of 42 μM⁻¹ s⁻¹.

The Binding of κ-Conotoxin PVIIA and Fast C-Type Inactivation of Shaker K+ Channels are Mutually Exclusive

Biophys J. Biophys J;86(1):191-209.