Contributed by Baldomero M. Olivera, November 16, 2018 (sent for review August 14, 2018; reviewed by Richard W. Aldrich and Michael M. Tamkun)
κM-RIIIJ blocks homomeric and heteromeric Kv1 channels. (A) Schematic representation of all possible combinations between two different Kv1 subunits. Homomeric and heteromeric channels containing Kv1.2 α-subunits and a second other Kv1 family (Kv1.x) subunit are displayed. Similar combinations are possible for all Kv1.x subunits with up to four different subunits forming a functional channel. (B) κM-RIIIJ block of homo- and heterodimeric Kv1 channels. Current traces in control (black) and in the presence of κM-RIIIJ (red) are shown. (Left) At 1 µM, κM-RIIIJ blocks homomeric Kv1.1 and Kv1.6 with low apparent affinity (IC50 ∼ 3–5 µM), whereas it displays ∼10× higher potency for Kv1.2 homomers (IC50 ∼ 300 nM). (Right) Representative dimeric channel-mediated currents in control and 100 nM κM-RIIIJ evidencing stronger block of Kv1.2/1.1 and Kv1.2/1.6 channels. Due to extreme differences in apparent affinity, 50 nM κM-RIIIJ is shown for Kv1.2/1.1 and 2 μM for Kv1.2/1.4. Dashed lines indicate zero current. (Scale bars, 500 pA and 10 ms.) (C) κM-RIIIJ affinity toward Kv1.2-containing dimeric Kv1 channels. The bar diagram presented summarizes κM-RIIIJ IC50s for dimeric constructs in forward or reversed order. In comparison with homomeric Kv1.2 channels, κM-RIIIJ has a 10- to 20-fold higher affinity for heterodimeric channels composed of Kv1.2 and either Kv1.1 or Kv1.6 subunits. All other dimeric constructs are blocked similarly or less strongly than homomeric Kv1.2. The order of the subunits in the concatemers bears no major influence on κM-RIIIJ’s activity.
κM-RIIIJ displays enhanced apparent affinity toward asymmetric Kv1 channels. (A) Western blot analysis of selected concatemeric constructs expressed in HEK293. Bands corresponding to 75-, 150-, and 300-kDa protein products were recognized by an anti-Kv1.2 antibody evidencing expression of monomeric, dimeric, and tetrameric channels, respectively. (B) Representative current traces of symmetric heterodimeric and asymmetric heterotetrameric channels composed of Kv1.2 and Kv1.6. The effect of 0.5 nM (red) and 5 nM (blue) κM-RIIIJ on evoked K+ currents from Kv1.6/1.2 dimer and tetrameric concatemers composed of three Kv1.2 subunits and one Kv1.6 subunit are shown (black: control). The zero current level is marked by the dashed line. (Scale bars, 1 nA and 50 ms.) (C) IC50s of κM-RIIIJ block of homomeric and heteroconcatemeric Kv1 channels formed by Kv1.1, Kv1.2, and Kv1.6 subunits in different combinations. κM-RIIIJ displayed extremely high apparent affinity (sub-nM) for asymmetric concatemers composed of one Kv1.1 or Kv1.6 subunit together with three Kv1.2 subunits (SI Appendix, Table S2).
Binding of κM-RIIIJ to the Kv1 channel pore. (A) Pore region sequence alignment of Shaker and Kv1 channel subunits. Observe that in Kv1.1 and Kv1.6 there is a tyrosine (Y379 and Y429, respectively, in red) homologous to Shaker T449. Identical amino acids to Shaker are shown as dots. Amino acids that differ from the majority are shaded in gray. (B) Tyrosine incorporation into a κM-RIIIJ–resistant dimer Kv1.2/1.4 renders it highly sensitive. The 10 nM κM-RIIIJ hardly affects Kv1.2/1.4 mediated currents (Top Left), whereas the same [κM-RIIIJ] leads to a profound block of Kv1.2/1.4-K531Y mediated currents (Top Right). Accordingly, Kv1.2/1.6 mediated currents are potently blocked by 10 nM of κM-RIIIJ, whereas sensitivity to the same [κM-RIIIJ] was abolished in the reciprocal mutant Kv1.2/1.6-Y429K. These results underscore the importance of the bulkier aromatic residue (tyrosine) at this position for κM-RIIIJ’s high-affinity binding. Zero current is indicated by the dashed line. [Scale bars, 2 nA (dimeric-WT), 200 pA (dimeric mutants), and 10 ms.] (C) Molecular dynamics simulation for κM-RIIIJ binding to Kv1.2/1.2/1.2/1.1 heterotetramer. Trajectories have been clustered to reveal different κM-RIIIJ docking positions. Visualization of the largest cluster (constituting 29% of the observed poses) is shown in more detail. Kv1.2 subunits are colored gray in cartoon representation, while the Kv1.1 subunit is black. κM-RIIIJ is represented by the green licorice and surface. κM-RIIIJ-Lys9 interacts with the channel selectivity filter (blue) occluding the permeation pathway, while Lys10 and Lys24 interact with Kv1.1-Tyr379 (homologous to Kv1.6-Tyr429).
κM-RIIIJ can distinguish between different sensory neuron subtypes. Representative calcium-imaging traces from three DRG neuronal subclasses displaying distinct responses to κM-RIIIJ and Dtx-K. (A) Neurons unaffected by κM-RIIIJ or Dtx-K: Four representative neurons unaltered by the application of κM-RIIIJ (25 nM, 75 nM, and 1 μM) and Dtx-K (100 nM), suggesting the lack of functional expression of Kv1.1 and Kv1.2 channels in these neurons. (B) Neurons responsive to the application of low [κM-RIIIJ]. The examples shown correspond to neurons that displayed an increase in peak height following a depolarizing stimulus in the presence of 25 nM, 75 nM, and 1 μM κM-RIIIJ and 100 nM Dtx-K, indicating the presence of both Kv1.1 and Kv1.2 channels in these neurons. (C) Neurons exclusively responsive to high [κM-RIIIJ]. These neurons were not affected by the application of low doses (25 and 75 nM) of κM-RIIIJ nor 100 nM Dtx-K but displayed increases in peak height after exposure to 1 μM κM-RIIIJ. The low sensitivity to κM-RIIIJ and insensitivity to Dtx-K suggest the presence of homotetrameric Kv1.2 channels and the absence of homomeric Kv1.1 or heteromeric Kv1.1/1.2 complexes in this cell population. Each trace corresponds to individual cells. The x axis is time (in minutes), and the y axis tracks the ratio of relative fluorescence change (ΔF/F) excited at 340 and 380 nM. Upward arrows mark the application of the depolarizing stimulus (20 mM [K+]o, ∼15 s). Colored boxes (red: κM-RIIIJ; blue: dendrotoxin-K) indicate the time of peptide exposure (∼6 min) in between the applications of depolarizing stimulus. Numbers to the right indicate cell area in μm2.
