Dynamics of internal pore opening in K(V) channels probed by a fluorescent unnatural amino acid

Abstract

Atomic-scale models on the gating mechanism of voltage-gated potassium channels (Kv) are based on linear interpolations between static structures of their initial and final state derived from crystallography and molecular dynamics simulations, and, thus, lack dynamic structural information. The lack of information on dynamics and intermediate states makes it difficult to associate the structural with the dynamic functional data obtained with electrophysiology. Although voltage-clamp fluorometry fills this gap, it is limited to sites extracellularly accessible, when the key region for gating is located at the cytosolic side of the channels. Here, we solved this problem by performing voltage-clamp fluorometry with a fluorescent unnatural amino acid. By using an orthogonal tRNA-synthetase pair, the fluorescent unnatural amino acid was incorporated in the Shaker voltage-gated potassium channel at key regions that were previously inaccessible. Thus, we defined which parts act independently and which parts act cooperatively and found pore opening to occur in two sequential transitions.

Keywords: Anap; two-color VCF.

Fig. 1.

(A) Structure of K_V1.2/2.1 chimera (PDB ID code 2R9R, two subunits) and topology (transmembrane segments S1–S6). The position of the mutations V234, A359, and H486 are displayed in green, and the potassium ions in the selectivity filter in red. (B) Principle of expression of fUAA. First, the plasmid to express the AnapRS and the corresponding tRNA is injected. On the subsequent day, fUAA and channel mRNA are injected, which leads to incorporation of the fUAA into the channel. (C) Structure of Anap. (D) Position of A359 (red) with respect to the arginines in the S4 (blue; PDB ID code 2A79). (E) Fluorescence response and gating currents of A359Anap in response to pulses from −90 mV to potentials between −180 and 50 mV. (F) Fluorescence voltage (FV, red circles), gating charge voltage (QV, black squares), and conductance voltage (GV, blue triangles) relations of A359Anap. The GV was fitted to a Boltzmann relation (V_1/2 = −29.1 mV, dV = 12.6 mV). The QV and FV were fitted to a sum of two (V_1/2,1 = −66.3 mV, dV₁ = 19.1 mV, V_1/2,2 = −35.2 mV, dV₂ = 5.4 mV) and three Boltzmann relations (V_1/2,1 = −134.6 mV, dV₁ = 32.8 mV, V_1/2,2 = −62.2mV, dV₂ =15.1 mV, V_1/2,3 = −41.6 mV, dV₃ = 11.6 mV), respectively.

Fig. 2.

(A) Gating currents and fluorescence responses of a TMR-labeled oocyte expressing V234Anap-A359C in response to pulses from −90 mV to potentials between −150 and 100 mV. (B) Anap fluorescence voltage (FV, red circles), gating charge voltage (QV, black squares) and conductance voltage (GV, blue triangles) relations of V234Anap-A359C. The GV and QV were each fitted to Boltzmann relations (GV: V_1/2 = 20.3 mV, dV = 20.6 mV; QV: V_1/2 = −61.4 mV, dV = 14.5 mV). The FV was fitted to a sum of two Boltzmann relations (V_1/2,1 = −56.7 mV, dV₁ = 11.8 mV, V_1/2,2 = −1.0 mV, dV₂ = 25.4 mV). F₁V refers to the first component of the FV. (C) Comparison of time dependence between charge movement (black) and fluorescence changes of V234Anap-A359C (red, Anap; green, TMR) for depolarizing pulses to voltages indicated in Inset. (D) TMR fluorescence voltage (FV, green diamonds) relation of V234Anap-A359C and the QV and GV with colors coded as in B. The FV was fitted to a sum of two Boltzmann relations (V_1/2,1 = −68.7 mV, dV₁ = 14.3 mV, V_1/2,2 = 0 mV, dV₂ = 23.0 mV). (E) Comparison among time constants of charge movement (black), Anap (red), and TMR (green) fluorescence of V234Anap-A359C. (F) Anap fluorescence voltage relation of the conducting mutant V234Anap. (G) Superposition of fluorescence traces obtained from a prolonged depolarizing pulse to 50 mV of oocytes expressing the conducting V234Anap mutant in absence (gray) or presence of 5 mM external 4-AP (red) and the nonconducting V234Anap mutant (black). The traces are normalized to the value at the end of the pulse. (Inset) Shown is the correlation between ionic current and fluorescence decay for the V234Anap-conducting mutant.

Fig. 3.

(A) Fluorescence response and gating currents of H486Anap in response to pulses from −90 mV to potentials between −140 and 150 mV. (B) Fluorescence voltage (FV, red circles), gating charge voltage (QV, black squares), and conductance voltage (GV, blue triangles) relations of H486Anap. QV and GV were fitted to Boltzmann relations (QV: V_1/2 = −47.2 mV, dV = 16.2 mV; GV: V_1/2 = 75.7mV, dV = 20.9 mV). The FV was fitted to a sum of two Boltzmann relations (V_1/2,1 = 0.5 mV, dV₁ = 14.2 mV, V_1/2,2 = 75.7 mV, dV₂ = 16.4 mV. (C) Comparison of time dependence between conductance (black) and fluorescence (red) of H486Anap. Fluorescence changes are observed in the absence of ion conduction and clearly precede ion conduction at −20 mV and +40 mV, respectively.