K(V)4.2 channels tagged in the S1-S2 loop for alpha-bungarotoxin binding provide a new tool for studies of channel expression and localization

Abstract

We report the first successful insertion of an engineered, high-affinity alpha-bungarotoxin (Bgtx) binding site into a voltage-gated ion channel, K(V)4.2, using a short, intra-protein embedded sequence (GGWRYYESSLEPYPDGG), derived from a previously described mimotope peptide, HAP. A major benefit to this approach is the ability to live-image the distribution and fate of functional channels on the plasma membrane surface. The Bgtx binding sequence was introduced into the putative extracellular loop between the S1 and S2 transmembrane domains of K(V)4.2. Following co-expression with KChIP3 in tsA201 cells, S1-S2 HAP-tagged channels express at levels comparable to wild-type K(V)4.2, and their activation and inactivation kinetics are minimally altered under most conditions. Binding assays, as well as live staining of surface-expressed K(V)4.2 channels with fluorescent-Bgtx, readily demonstrate specific binding of Bgtx to HAP-tagged K(V)4.2 expressed on the surface of tsA201 cells. Similar live-imaging results were obtained with HAP-tagged K(V)4.2 transfected into hippocampal neurons in primary culture suggesting applicability for future in vivo studies. Furthermore, the activation kinetics of S1-S2-tagged K(V)4.2 channels are minimally affected by the binding of Bgtx, suggesting a limited role if any for the S1-S2 loop in voltage sensing or gating associated conformational changes. Successful functional insertion of the HAP sequence into the S1-S2 linker of K(V)4.2 suggests that other related channels may similarly be amenable to this tagging strategy.

Figure 1

Schematic representation of the insertion of the HAP and Tα1 tag into the S1–S2 linker of Kv4.2. The diagrams depicting the structure of the neuronal nicotinic receptor (left) next to the structure of alpha-Bgtx (right) and the topology of Kv4.2 are shown. Two engineered sequences were inserted individually into the extracellular S1–S2 loop. One is derived from the principal Bgtx binding site on the Torpedo nAChR α1 subunit (Tα1) with sequence of WVYYTCCPDTPYLD. The other is from a high affinity peptide (HAP) sequence, WRYYESSLLPYPD, derived from a combinatorial phage-displayed library. These sequences were inserted into a location distant from the voltage sensor (S4) and ion selectivity filter (P loop) to minimize perturbation of the channel structure and function.

Figure 2

Heterologous expression of wild-type Kv4.2, HAP-tagged Kv4.2 (Kv4.2-HAP) and EGFP plus HAP–tagged Kv4.2 (Kv4.2-HAP-EGFP) in tsA201 cells. (a) K+ currents in tsA201 cells were elicited by voltage clamp steps delivered at 10-mV increments from a holding potential of −80 mV to step depolarization from −30 mV to +50 mV (a, inset). Each voltage step was 600 ms long. Representative traces are from mock-transfected cells, or cells transfected with Kv4.2, Kv4.2-HAP, or Kv4.2-EGFP-HAP as indicated. (b) Western blotting of Kv4.2 vs. Kv4.2-HAP and Kv4.2-HAP-EGFP vs. Kv4.2-EGFP. All constructs were detected with anti-Kv4.2 antibody.

Figure 3

Activation kinetics for Kv4.2 (■) and Kv4.2-HAP (). (a) Time to peak current analysis. Representative current traces of inactivation for Kv4.2 and Kv4.2-HAP are shown in Fig1(a). The upper panel shows detailed superimposed peak currents for Kv4.2 (black) and Kv4.2-HAP (red) in response to step membrane depolarization. Student’s t test analysis showed that for voltage steps +20 mV to +50 mV the mean times to peak for Kv4.2 and Kv4.2-HAP were not significantly different (p > 0.05). In contrast, for the −30 to +10 mV range, the results are significantly different (p < 0.05). (b) Normalized peak conductance-voltage relations for Kv4.2 and Kv4.2-HAP. Normalized conductance (G/G_max, conductances at the indicated potential divided by G_max, the maximum conductance, at +50 mV) plotted as a function of voltage for the currents expressed by Kv4.2 and Kv4.2-HAP. Peak conductance (G) was calculated as G = I_p/(V_m − V_eq), where I_p, V_m and V_eq are the peak current, the test potential and the K+ equilibrium potential, respectively. The continuous lines across the data points are the best-fits to Boltzmann functions. Values represent the Mean ± S.E.M of three to four cells. Normalized peak conductance-voltage relations for Kv4.2 and Kv4.2-HAP at all voltage steps are not significantly different by the Student’s t test (p > 0.05).

Figure 4

Inactivation time constants τ Kv4.2 (■) and τ Kv4.2-HAP (). The upper panel shows inactivation kinetics of currents in response to step membrane depolarization. The time course of fast inactivation fits mono-exponentially. The continuous lines in red overlaid represent single exponential fits. Note that, the rate of inactivation increases with strong depolarization. Rate of inactivation decreased in the voltage range from −30 mV to 0 mV and increased from 0 mV to +50 mV. Values represent the Mean ± S.E.M of three to four cells. Student’s t test analysis showed that the means of inactivation time constants τ for Kv4.2 and Kv4.2-HAP at voltage steps ranging from −20 mV to +20 mV are not significantly different (p > 0.05). In contrast, they are significantly different at voltage steps of −30 mV, +30 mV, +40 mV and +50 mV (p < 0.05).

Figure 5

Rate of recovery from inactivation. (a) Representative current traces of the recovery from inactivation for Kv4.2, Kv4.2-HAP and Kv4.2-EGFP-HAP respectively are shown. A paired pulse protocol shown as an insert was applied to transfected tsA201 cells. Each cell was depolarized from −80 to +50 mV by two steps, varying the inter-step intervals (Δt) from 5 ms to 245 ms in 10 ms increments. The membrane potential during the interval was also −80 mV. This set of paired pulses was applied once every 15 s. (b) The time course of recovery from inactivation is analyzed by normalizing the peak current amplitude of the second pulse to that of the first pulse and plotting as a function of inter-pulse duration. Kv4.2-HAP () recovers slightly more slowly from inactivation than wild-type Kv4.2 (■). Values represent the Mean ± S.E.M of data obtained from three to four cells. Applying the Student’s t test, the rate of recovery from inactivation for Kv4.2-HAP is not significantly different (p > 0.05) from that of Kv4.2.

Figure 6

Binding of ¹²⁵I -Bgtx to Kv4.2-HAP and effect of Bgtx on Kv4.2-HAP currents. (A) Binding of ¹²⁵I -Bgtx to Kv4.2-HAP. tsA201 cells were incubated with ¹²⁵I for 2 h. Specific Bgtx binding was observed for Kv4.2-HAP as well as muscle-type nAChR (p < 0.05; denoted by asterisk,*), but not for Kv4.2 or Kv4.2-Tα1. Values represent the Mean ± S.E.M. The data shown are representative of three experiments. (B) Effect of Bgtx on Kv4.2-HAP currents. The current traces were recorded in the absence (black) or in the presence of 100 nM Bgtx (red). The presence of Bgtx had little effect on the activation of the channels.

Figure 7

Alexa Fluor-Bgtx binds Kv4.2-HAP expressed in tsA201 cells. DIC, image of differential interference contrast; GFP, fluorescent image of GFP (detecting co-transfected GFP, green); Alexa Fluor-Bgtx, fluorescent image of Alexa Fluor-Bgtx (detecting Kv4.2-HAP, red). Wild-type Kv4.2 or HAP-tagged Kv4.2 was co-transfected with GFP in tsA201 cells. Fluorescent Bgtx bound HAP-tagged Kv4.2 and was competed off in the presence of excess unlabeled Bgtx. No fluorescent Bgtx binding was observed for wild-type Kv4.2.

Figure 8

Rhodamine-Bgtx binds Kv4.2-HAP-GFP and GluR2-HAP-GFP expressed in primary rat hippocampal neurons. (A) A neuron transfected with Kv4.2-HAP-GFP. Bright Field, bright-field image of the neuron; GFP, fluorescence image of GFP (detecting all Kv4.2-HAP-GFP channels); Rhod.-Bgtx, fluorescence image of rhodamine-Bgtx (detecting membrane-inserted Kv4.2-HAP-GFP). (B) Cumulative curves and histograms of Kv4.2-HAP-GFP cluster size distribution obtained for green and red clusters (n=6 cells). (C) A neuron transfected with GluR2-HAP-GFP. Bright Field, bright-field image of the neuron; GFP, fluorescence image of GFP (detecting both perimembrane and membrane-inserted GluR2-HAP-GFP channels); Rhod.-Bgtx, fluorescence image of rhodamine-Bgtx (detecting only membrane-inserted GluR2-HAPGFP). (D) Cumulative curves and histograms of GluR2-HAP-GFP cluster size distribution obtained for green and red clusters (n=3 cells). Scale bars: 5 µm.