Portability of paddle motif function and pharmacology in voltage sensors

Abstract

Voltage-sensing domains enable membrane proteins to sense and react to changes in membrane voltage. Although identifiable S1-S4 voltage-sensing domains are found in an array of conventional ion channels and in other membrane proteins that lack pore domains, the extent to which their voltage-sensing mechanisms are conserved is unknown. Here we show that the voltage-sensor paddle, a motif composed of S3b and S4 helices, can drive channel opening with membrane depolarization when transplanted from an archaebacterial voltage-activated potassium channel (KvAP) or voltage-sensing domain proteins (Hv1 and Ci-VSP) into eukaryotic voltage-activated potassium channels. Tarantula toxins that partition into membranes can interact with these paddle motifs at the protein-lipid interface and similarly perturb voltage-sensor activation in both ion channels and proteins with a voltage-sensing domain. Our results show that paddle motifs are modular, that their functions are conserved in voltage sensors, and that they move in the relatively unconstrained environment of the lipid membrane. The widespread targeting of voltage-sensor paddles by toxins demonstrates that this modular structural motif is an important pharmacological target.

Figure 1. Transfer of the voltage-sensor paddle motif from KvAP to Kv2.1 channels

a, Overview of chimeras between KvAP (blue) and Kv2.1 (black). Constructs that result in functional Kv channel activity when expressed in oocytes are indicated with green circles and those that are non-functional are indicated with red circles. Chimera nomenclature is Cx[region transferred]donor channel, where x is the chimera number within that region. b, Defining the region within the voltage sensor of KvAP (indicated by dashed lines) that results in functional channels when transferred to Kv2.1. Alignment between KvAP and Kv2.1 in S3 through S4, highlighting (blue bar) the stretch of residues transferred to form C7[S3–S4]AP. Conserved residues are shown in bold lettering. c, Backbone fold of a single subunit from KvAP (PDB: 2A0L) (left) depicting the paddle region in blue. Arrow indicates the permeation pathway for potassium ions. These and all subsequent structures were created using PyMOL (DeLano Scientific LLC). d, Potassium currents and tail current voltage-activation relations (n = 5–12; error bars are S.E.M.) for Kv2.1 and the C7[S3–S4]AP paddle chimera after expression in oocytes. Holding voltage was −80 mV and tail voltage was −60 mV.

Figure 2. Sensitivity of KvAP paddle chimeras to extracellular tarantula toxins

a, Channel constructs, designated at the left with KvAP segments shown in blue, were expressed in oocytes and potassium currents elicited by depolarizations in the absence (black) or presence (red) of either HaTx or VSTx1. Depolarizations were to voltages near the foot of the voltage-activation relationship (relative open probability < 0.3) for each construct. All chimeras involving Kv2.1 are defined in Supplementary Fig 1. The paddle chimera in Shaker (C*[S3–S4]AP) was generated by transplanting P99–R126 of KvAP into P322–R371 of Shaker. C*[S3–S4]AP was studied with a low K⁺ external solution and all others were studied with a high K⁺ external solution (see Methods). Shaker has a very low sensitivity to HaTx and was not studied. VSTx1-insensitive channels were only studied at the highest VSTx concentration. b, VSTx1 inhibition of chimera C2[S4]AP is voltage-dependent. Potassium currents were recorded for weak (0 mV, left) and strong (60 mV, right) depolarizations, before and after addition of 12 µM VSTx1. Inset to the far right shows scaled tail currents after depolarization to 60 mV. c, Families of currents recorded in response to depolarizations in the absence (black) and presence of 12 µM VSTx1 (red). Holding voltage was −90 mV and tail voltage was −60 mV. Corresponding tail current voltage-activation relations for the traces shown, where tail current amplitude is plotted against test voltage.

Figure 3. Structural analysis of the toxin-paddle interaction

a, Alanine scan of the paddle motif of KvAP in the C*[S3–S4]AP chimera where perturbations in apparent VSTx1 affinity (K_d^mut/K_d^cont) are plotted for individual Ala and Val mutants. The dashed line marks a value of 1 and numbering corresponds to the amino acid sequence of KvAP. Each mutant was initially examined using a concentration of toxin near the K_d for the control chimera; mutants displaying higher K_d values were further examined using higher toxin concentrations. n = 3–5 for each toxin concentration and error bars are S.E.M.. b, Stereo pair of the isolated voltage-sensing domain of KvAP (PDB: 1ORS) with side chains in the paddle colored according to perturbations in toxin affinity as in a. c, Positioning of the voltage-sensing domain of KvAP adjacent to a hypothetical pore domain according to the X-ray structure of Kv1.2 (PDB: 2A79). The α-carbon of G108 is indicated by a red asterisk.

Figure 4. Transfer of the voltage-sensor paddle motif from Hv1 or Ci-VSP into Kv2.1 channels

Overview of chimeras between Hv1 (a, light blue), Ci-VSP (b, dark blue) and Kv2.1 (black). Constructs that result in functional Kv channel activity when expressed in oocytes are indicated with green circles and those that are non-functional are indicated with red circles. Dashed lines are the same as in Fig 1. The amino acid alignment shows the sequence of Kv2.1, Hv1 and Ci-VSP in S3 through S4, highlighting (blue bars) the stretch of residues transferred to form the two chimeras indicated. c, Current traces and tail current voltage-activation relations (n = 3; error bars are S.E.M.) for a chimera expressed in oocytes where the paddle of Ci-VSP was transferred into Kv2.1. Test depolarizations were to voltages between −50 and +80 mV, holding voltage was −80 mV and tail voltage was −50 mV. d, Current traces and tail current voltage-activation relations (n = 3; error bars are S.E.M.) for a chimera where the paddle of Hv1 was transferred into Kv2.1. Test depolarizations were to voltages between −100 and +40 mV, holding voltage was −90 mV and tail voltage was −90 mV.

Figure 5. Sensitivity of a Hv1 paddle chimera and the Hv1 proton channel to tarantula toxins

a, Voltage-activation relations in the absence (black) and presence (red) of tarantula venom/toxins for a chimera containing the paddle of Hv1 in the Kv2.1 channel. Potassium currents were recorded using 300–500 ms test depolarizations from holding voltages between −100 and −90 mV, and tail voltages between −100 and −90 mV. Venom was applied at a 1:5000 dilution and toxin concentrations (in µM) were 2 for HaTx, 8 for VSTx1, 4 for SGTx1, 1 for GxTx1E and 10 for GmTxSIA. b, Proton currents recorded for Hv1 in response to weak (+30 mV; top) and strong (+60 mV; bottom) depolarizations, both in the absence (black) and presence (red) of 4 µM HaTx. Hv1 was expressed in HEK cells. Holding voltage was −40 mV and tail voltage was −60 mV. b, Tail current voltage-activation relations for Hv1 recorded in the absence (black) and presence of 1 µM (red squares) or 4 µM (red diamonds) HaTx. For all voltage-activation relations n = 4–5 and error bars are S.E.M..

Figure 6. Tarantula toxins interacting with voltage-sensor paddle motifs

Cartoon depicting the interaction of tarantula toxins with voltage-sensor paddle motifs within the lipid membrane for Kv channels, and the voltage-sensing domain proteins, Ci-VSP and Hv1. Only one of the four voltage-sensing domains is shown surrounding the pore domain in Kv channels.