Binding kinetics of quaternary ammonium ions in Kcv potassium channels

Abstract

Kcv channels from plant viruses represent the autonomous pore module of potassium channels, devoid of any regulatory domains. These small proteins show very reproducible single-channel behavior in planar lipid bilayers. Thus, they are an optimum system for the study of the biophysics of ion transport and gating. Structural models based on homology modeling have been used successfully, but experimental structural data are currently not available. Here we determine the size of the cytosolic pore entrance by studying the blocker kinetics. Blocker binding and dissociation rate constants ranging from 0.01 to 1000 ms^-1 were determined for different quaternary ammonium ions. We found that the cytosolic pore entrance of Kcv_NTS must be at least 11 Å wide. The results further indicate that the residues controlling a cytosolic gate in one of the Kcv isoforms influence blocker binding/dissociation as well as a second gate even when the cytosolic gate is in the open state. The voltage dependence of the rate constant of blocker release is used to test, which blockers bind to the same binding site.

Keywords: Fast block; blocker; diffusion limitation; planar lipid bilayer; single-channel currents; viral potassium channel.

Figure 1.

Markov models used for the fitting of the blocking kinetics. (a) The full Markov model with all states as known so far from previous studies [2,3,29] supplemented by the blocked state B. The states C₁, C₂ and C₃ belong to slow gating with dwell times longer than a millisecond, with C₃ occurring only in Kcv_S due to its cytosolic gate [2]. These states can be analyzed by a jump detector and dwell time analysis. F and M belong to fast gating (dwell-time in F about 5 µs) and medium gating (dwell-time in M is voltage-dependent ranging between 150 µs to 40 µs) [3]. (b, c) The models used for the analysis of fast blockers. C₁ - C₃ cannot be resolved by the beta distribution analysis and are merged into S (“slow”). (c) For TEA, B has a similar dwell time as F, so the two states cannot be kinetically separated. (b) for TPrA, this separation is possible [7]. (d,e,f) in the experiments with slow blockers, the dwell time in B coincides with one of the slow states C₁ - C₃. Thus, only two (Kcv_NTS) or three (Kcv_S) closed states are detected in the dwell time histograms. The inclusion of B is identified by concentration dependence. O,F,M are merged into an apparent open state O*. Definition: The rate constant of the transition from a state X to a state Y is called k_XY. k_BO as the rate constant of blocker dissociation is also called k_off.

Figure 2.

(a) Comparison of the structure of a homology model for Kcv_NTS and the structure of the TBA@KcsA complex (pdb code 2HVK), which is used as an illustration of the location of the blocker [41]. Two monomers of the KcsA channel are shown in gray, the TBA ion (yellow) is located inside the cavity. The homology model of Kcv_NTS on the template structure of KirBac1.1 (1P7B) calculated with Swissmodel [42] is shown in black with the amino acids discussed in this work highlighted in orange. Structures drawn with UCSF chimera [43]. (b) size of quaternary ammonium blockers (QAs), as estimated by [44]. The colors of the alkyl chains refer to the circles with the same color. The same accessible color palette ([45] and https://www.accessiblecolorpalette.com/, accessed June 2024) for the blockers is used in all figures in this work. (c) Sequence alignment of Kcv_NTS and Kcv_S. Residues I73, G/S77 and F78 are highlighted in orange. (d) Representative single-channel traces of Kcv_NTS and Kcv_S at +120 mV in a DPhPC membrane in 100 mM KCl.

Figure 3.

Blocking properties of Kcv_NTS by cytosolic QAs. (a) Single-channel fluctuations in DPhPC bilayers in symmetric 100 mM KCl without and with different QAs recorded at +120 mV (upper traces) and −120 mV (lower traces). The closed channel is indicated by a dashed line. The blocker concentrations are given above the traces. (b) Apparent single-channel channel I_app/V-relations without (black) and with (colored) blocker, concentrations indicated at the curves in either (b) or (c). (c) Apparent open probabilities and (d) time averaged I_avg/V relations with I_avg = I_app·P₀. Mean ± s.D., number of independent recordings is indicated in brackets.

Figure 4.

Representative concentration response curves of inhibition Inh_exp for (a,b) TPrA (9 Å diameter) and (c,d) TPeA (10.5 Å) in (a,c) Kcv_NTS and in (b,d) Kcv_S as obtained from the data in Figure 3 and Figure S2, respectively. Fit with Eq. 5. (e) Voltage dependence of apparent K_D (as obtained from fits like those in Figure 4a–d) for the different blockers in Kcv_NTS (filled circles) and Kcv_S (open circles). TMA: purple, TEA: green, TPrA: dark blue, TBA: yellow, TPeA: light blue, THxA: orange. *: At these voltages, the K_D for TPrA and TBA differ significantly from each other in Kcv_NTS and Kcv_S. “n.s.:” The K_D for TPeA (Kcv_NTS and Kcv_S) and TPeA (Kcv_NTS) do not differ significantly at any voltage. One-parameter ANOVA with threshold p = 0.05.

Figure 5.

Kinetics of the open-state block with TPrA for the two channel isoforms Kcv_NTS (filled symbols, no cytosolic gate) and Kcv_S open symbols, with cytosolic gate) (Figure 2a,c). (a) Rate constant k_OB of blocker binding and (b) rate constant k_BO = k_off of blocker release for 0.1 mM cytosolic TPrA. Averages ± sd of three experiments each. The small errors are a typical benefit of Kcv channels.

Figure 6.

Dwell-time histograms of (a,c) Kcv_NTS and (b,d) Kcv_S under control conditions and blocked by TPeA. Exemplary closed- and open-time histograms at +100 mV for the control (a,b) and 200 nM TPeA (c,d). Open histograms (right hand side in each panel) were fitted by Eq. 1 (black) with a single exponential. Closed time histograms were fitted with a sum (black, continuous line) of two exponentials for Kcv_NTS or three exponentials for Kcv_S. Individual components: C₁ = dotted, C₂ = dashed, C₃ = dash-dot. Inclusion of state B in one of the closed states is indicated by the arrows.

Figure 7.

Dependence of occupation probabilities P of the states in the Markov models of Figure 1e or f on TPeA concentration. Association of the blocked state B to C₂ (diamonds in (a-c)) or C₃ (crosses in (d-f) is based on the dependence of their occupation probabilities on blocker concentration. The occupation probabilities of the open state (P_O, circles) and of the two or three closed states (P_C1, triangles, P_C2, diamonds, and P_C3, crosses) are shown for (a,b,c) Kcv_NTS (filled symbols) and for (d,e,f) Kcv_S (open symbols) at different TPeA concentrations at (a,d) +100 mV, (b,e) +60 mV and (c,f) −60 mV. Averages ± sd of three experiments each. Orange symbols in the curves related to B mark the concentrations, where the occupation probability of the respective state exceeds 10 times that of the control value and are therefore dominated by B. Grey symbols indicate that this probability is too small for the identification of B. for Kcv_S at −60 mV, and for both channels at −100 mV (not shown), this threshold was not reached (only gray symbols). The two additional gray triangles in B are data points, where due to the low probability of C₁, n = 3 could not be achieved for this state. This did not affect the determination of C₂.

Figure 8.

Rate constants of blocking of the open channel (a,c,e) k_OB and (b,d,f) k_BO as obtained from the dwell time histograms in Figure 6 by means of Eqs. 1 and 3 at −60 mV, +60 mV and +100 mV. Filled symbols: Kcv_NTS, open symbols; Kcv_S. Orange symbols are those where the blocked state B dominates the intrinsic closed state, and the rate constants can be taken as a good approximation of the rate constants of blocking. Grey symbols: this condition was not met; the rate constant was not used for further analysis. Mean ± sd of three experiments each.

Figure 9.

(a,b) Voltage dependence of the rate constants of open-state block (A) k_on as calculated by Eq. 6 and (B) k_off for different QAs. (c,d) Dependence of (C) k_on and (D) k_off at +100 mV on blocker diameter estimated by Robinson and Stokes [46]. Filled symbols: Kcv_NTS, open symbols: Kcv_S. Colors are defined in (A). Averages +/- sd of three experiments each.

Figure 10.

(a) Exemplary fit of k_off on an Kcv_NTS data set with 0.5 mM cytosolic TPrA with the equation k_off = k_off,0·exp(-zδ·FV/RT), zδ = 0.28, k_off,0 = 1.05·10⁵ s^−1. (b) Overlay of the normalized (see text) k_off for TPrA (dark blue), TBA (yellow), TPrA (orange) and THxA (light blue) in Kcv_NTS. §: No significant difference between the data points at this voltage. *: at least one data point is significantly different from the others. (c) Normalized (see text) K_D of all blockers in Kcv_NTS. A significant difference for at least one of the data points from the others is only found at +40 mV. One-parameter ANOVA with threshold p = 0.05.

Figure 11.

Block by TPrA. Influence of the residues at positions 77 and 78 on the rate constants of (a, b, c) blocker binding k_on = k_OB/[TPrA] and (d,e,f) release (k_BO = k_off) in different mutants of Kcv_NTS and of Kcv_S. Lines show the wild-type data (Kcv_NTS: continuous, Kcv_S: dashed), the symbols indicate the residue at locations 77: G = filled, S = open for the mutants (a,d) S77G and G77S (b,e) F78A and (c,f) the double mutants. “pooled:” wt data averaged over TPrA concentration from 0.05 – 5 mM (Kcv_NTS) and 0.1 – 1 mM (Kcv_S), data for Kcv_NTS S77G F78A averaged over 0.1 & 1 mM. All other blocker concentrations are indicated in the legends. All data points: mean ± sd of at least three experiments each. Because of the decrease of the signal with higher negative voltages, rate constants could not be determined from fitting amplitude histograms for voltages more negative than −80 mV or −60 mV for all mutants and Kcv_S.