Allosteric block of KCa2 channels by apamin

Abstract

Activation of small conductance calcium-activated potassium (K(Ca)2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin apamin, but the mechanism of block is not understood. For example, apamin binds to both K(Ca)2.2 and K(Ca)2.3 with the same high affinity (K(D) approximately 5 pM for both subtypes) but requires significantly higher concentrations to block functional current (IC(50) values of approximately 100 pM and approximately 5 nM, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that apamin blocks K(Ca)2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.

FIGURE 1.

Binding and block of K_Ca2.2 and K_Ca2.3 channel current by apamin. A and B, representative examples of saturation relationships for ¹²⁵I-apamin binding to expressed K_Ca2.2 (A) or K_Ca2.3 (B) subunits. C and D, whole-cell macroscopic currents derived from ramps from −80 to 80 mV imposed on voltage-clamped HEK293 cells expressing K_Ca2.2 (C) and (D) K_Ca2.3 subunits. The application of increasing concentrations of apamin inhibited macroscopic current. E, concentration-inhibition relationships for apamin inhibition of expressed K_Ca2.2 and K_Ca2.3 current.

FIGURE 2.

His-337/His-491 residues are critical for block of K_Ca2.2 and K_Ca2.3 currents by apamin and organic blockers. A–C, outside-out patch (A and C) and whole-cell (B) macroscopic currents evoked by ramps from −80 to 80 mV in the absence (black trace) and the presence of a supramaximal concentration of apamin (100 nm) (gray trace). Mutation of the outer pore His residue in K_Ca2.2(H337N) (A) and K_Ca2.3(H491N) (B) produced currents that were insensitive to the bee venom toxin. Mutation of His-337 in K_Ca2.2 to the positively charged arginine (H337R) also produced currents that were apamin-insensitive (C). D, graph showing the mean ± S.E. inhibition by 100 nm apamin of macroscopic current from each mutant. E, graph showing the lack of block of K_Ca2.2(H337N) by the organic blockers UCL1684 (20 nm) and d-TC (100 μm) when compared with block of WT K_Ca2.2- and K_Ca2.3-mediated current.

FIGURE 3.

Block of K_Ca2.2 and K_Ca2.2(H337N) channels by extracellular TEA. A and B, representative traces from outside-out patches of expressed K_Ca2.2 (A) and K_Ca2.2(H337N) (B) channel currents evoked by voltage ramps from −100 to 100 mV in the absence and presence of increasing concentrations of extracellular TEA. Experiments were performed in low (5 mm) extracellular [K⁺], with increasing concentrations of TEA being substituted for NaCl. C, concentration-inhibition relationships for block of wild-type K_Ca2.2 and mutant K_Ca2.2(H337N) current by TEA (see “Results” for details).

FIGURE 4.

Structural modeling of the interaction between the K_Ca2.2 channel and apamin or TEA. A and B, top down (A) and side (B)views of apamin docked to the outer pore region of K_Ca2.2. Channel residues discussed under “Results” are highlighted as follows: His-337 (yellow), Gln-339 (orange), Asn-345 (green), and Asn-368 (cyan). Residues within the channel outer pore and apamin that make contact by hydrogen bonds are colored blue, whereas those channel residues making electrostatic interactions are in red. C and D, top down (C) and side (D) views of the interaction between TEA and K_Ca2.2. The quaternary ammonium ion is modeled to interact within the inner pore of the channel by electrostatic interactions with the C=O group of Tyr-362 (distance between C=O and N⁺ ∼4 Å), and the ethyl groups of TEA interact via van der Waals contacts with Gly-363 (orange), Asp-364 (yellow), and Val-366 (cyan).

FIGURE 5.

Reduced sensitivity to block by apamin displayed by K_Ca2.2(N345G). A, concentration-inhibition relationship for block of expressed K_Ca2.2(N345G) channel current by apamin. The dashed gray curve shows the sensitivity of block of wild-type K_Ca2.2 current for comparison. B, concentration-inhibition relationship for block of K_Ca2.2(N345G) current by extracellular TEA. The relationship of block by TEA of wild-type K_Ca2.2 current is shown for comparison (dashed gray line), showing that the channel mutation had little effect upon block by the quaternary ion.

FIGURE 6.

Apamin and TEA block K_Ca2.3 current by using non-interacting binding sites. A, example trace of whole-cell holding current recorded at −80 mV from a cell expressing K_Ca2.3 channels and bathed in high extracellular K⁺ solution. Fast application of apamin (3 nm) produced a block with a τ_on of 0.70 ± 0.12 s. Fits are shown as gray lines in all panels. B, membrane current recorded at −50 mV from a cell expressing K_Ca2.3 channel subunits. Fast application of TEA (1.8 mm) blocked ∼30% of current but had no effect on the time course of subsequent block by fast applied apamin (3 nm)(τ_on of 0.79 ± 0.08 s). C, fast application of NML (5 μm) blocked ∼40% of expressed K_Ca2.3 current, with the presence of this channel blocker slowing the rate of block by the subsequent fast application of apamin (τ_on slowed to 1.36 ± 0.08 s). D, a greater slowing of the rate of block by apamin was produced by a higher concentration of NML, with τ_on of block by apamin (3 nm) being slowed to 1.84 ± 0.09 s in 7.5 μm NML. E, graph showing individual values from all experiments of the τ_on (Tau) of block by apamin (3 nm) applied in the absence or presence of either TEA (1.8 mm) or NML (5 and 7.5 μm). A one-way analysis of variance showed that τ_on values were significantly different from each other (F = 37, p < 0.001). Tukey's post-hoc tests showed that the τ_on of apamin was unaffected by TEA (p > 0.05), but was significantly affected by both concentrations of NML (p < 0.05). n. s., not significant. ***, significant with p < 0.001.

FIGURE 7.

Pore-mimicking mutants reveal differences between K_Ca2.2 and K_Ca2.3 structure. A, sequence alignment of the pore regions of K_Ca2.2 and K_Ca2.3 channels, with residues that differ highlighted in gray. SF, channel selectivity filter. B, concentration-inhibition relationships of K_Ca2.2(N368H), a mutation that produced a K_Ca2.2 channel whose pore mimicked K_Ca2.3. This mutation produced a channel current that was blocked by apamin with a sensitivity that was similar to that seen with wild-type K_Ca2.3 current. The relationships of block by apamin of wild-type K_Ca2.2 and K_Ca2.3 currents are shown for comparison in dashed gray. C, mutation of the outer pore of K_Ca2.3 to mimic K_Ca2.2 (K_Ca2.3(H522N)) produced a current that was blocked by apamin with a sensitivity that was not significantly different from wild-type K_Ca2.3 current. The relationships of block by apamin of wild-type K_Ca2.2 and K_Ca2.3 currents are shown for comparison in dashed gray. D, bar chart displaying the IC₅₀ values showing the reduction in sensitivity in K_Ca2.2(N368H) when compared with WT K_Ca2.2(p < 0.0001, n = 7–10). No significant shift was observed for K_Ca2.3(H522N) when compared with WT K_Ca2.3 (p > 0.05, n = 8–12).

FIGURE 8.

Co-expression of apamin-sensitive wild-type K_Ca2.2 and apamin-insensitive K_Ca2.2(H337N) produced heteromeric channels that displayed distinct sensitivities to apamin. A, representative outside-out patch current traces evoked by voltage ramps from −100 to 100 mV, in the absence (con, control) and presence of increasing concentrations of apamin. B, concentration-inhibition relationship for block of current produced by co-expression of wild-type K_Ca2.2 and mutant K_Ca2.2(H337N) channels by apamin. Mean data were fit with a two component Hill equation, with IC₅₀ values of ∼270 pm and 33 nm, demonstrating that heteromeric channels were expressed. frac, fraction. C, example trace of outside-out patch holding current (V_h −80 mV) from a cell co-expressing wild-type and mutant K_Ca2.2 channels during the rapid application and removal of apamin (3 nm). Current was blocked by apamin with an exponential time course (not shown), with the recovery from block being best described by the sum of two exponential components of taus: τ_off,1, 1.8 s, and τ_off,2, 36.4 s. These data suggest that apamin is interacting with two populations of channels that possess different sensitivities to block by the toxin. D, the probabilities of occurrence (P_occ) of different predicted stoichiometries of channel subunit assembly were calculated assuming that the probability of the inclusion of K_Ca2.2 and K_Ca2.2(H337N) subunits into the channel tetramer was equivalent.