Extracellular redox sensitivity of Kv1.2 potassium channels

Figures

• 

  Figure 1. Reducing conditions promote use-dependent activation. Conductance-voltage relationships were recorded from tail current amplitudes at −30 mV (see inset) in ltk- fibroblast cells expressing Kv1.2. Grey lines are conductance-voltage relationships from multiple individual cells in ambient redox conditions (spread of V1/2 is from −1.7 to +43 mV). Mean conductance-voltage relationships (±S.D.) are shown for cells incubated in 666 µM DTT (N = 13, blue symbols, V1/2 = +64 ± 11 mV), or collected with a modified protocol that delivers a 500 ms depolarization to +60 mV before each voltage sweep (black symbols, N = 11, V1/2 = −11 ± 3 mV). (Inset) Currents elicited with a + 40 mV depolarization illustrate the suppression of current in DTT. (B,i–iii) Kv1.2 expressing cells were stimulated with repetitive 10 ms depolarizations from a holding potential of −80 mV to + 60 mV (20 Hz frequency). Different sample sweeps reflect the variability of use-dependence in ambient redox (i,ii), and a shift towards strong use-dependence after incubation in DTT (iii). (C) % use-dependent activation (UDA) is calculated as the fraction of activating current during the pulse train: (pulse 100 peak current – pulse…

• 

  Figure 2. Time dependent recovery of inhibited gating mode. (A) Cells expressing Kv1.2 were stimulated with a train of repetitive depolarizations to +60 mV, followed by a sequence of 15 ms pulses to −10 mV (0.5 Hz) to assess recovery of the inhibited gating mode (detailed protocol in Supplemental Figure 2). Mean normalized test pulse magnitude (±S.D) for ambient redox (grey, time constant = 5.1 ± 2.9 s, N = 51) and DTT incubation (blue, time constant = 2.2 ± 1.0 s, N = 46) are presented, along with surfaces illustrating the range of variability observed in individual cells. (B) Cell-by-cell correlation of % use-dependent activation and % recovery of inhibition. Each data point reflects an individual cell (grey: ambient redox, blue: 666 µM DTT).

• 

  Figure 3. Redox-sensitive use-dependence is transferrable in heteromeric channels containing Kv1.2 subunits. (A) Repetitive depolarizations were used to assess % use-dependent activation, as described in Fig. 1B, for a variety of Kv1 channel subtypes and concatenated combinations with Kv1.2 as indicated. Each data point reflects the % use-dependence recorded from an individual cell in ambient redox conditions (gray) or in the presence of either 666 µM or 2 mM DTT (blue). N = 10–17 for each construct and condition. (B) Exemplar current tracings illustrating use-dependent activation of Kv1.1-Kv1.2 dimeric channels in (i) ambient redox conditions or (ii) 666 µM DTT.

• 

  Figure 4. Extracellular redox environment modulates Kv1.2 use-dependent activation. (A) Use-dependent activation was assessed using repetitive depolarizations as described in Fig. 1. DTT (membrane permeant, blue) or TCEP (membrane impermeant, orange) reducing agents were applied intracellularly at 500 µM (through the pipette solution) or extracellularly at 200 µM, as indicated (External agents: N = 41 for ambient redox, N = 12 for DTT and N = 15 for TCEP; internal agents: N = 12 for ambient redox, DTT and TCEP). (B) Exemplar current traces from cells expressing Kv1.2 channels in various reducing agents illustrate the membrane sidedness of TCEP effects. (C) Mouse ltk- fibroblasts transfected with the genetically encoded redox sensor Grx1-roGFP were perfused with a range of redox active compounds, as indicated (H2O2 at 1 mM, DTT and TCEP at 666 µM). Extracellular TCEP provides minimal recovery of intracellular redox potential, while DTT rapidly reduces the intracellular compartment (black symbols = mean ± S.D., grey symbols are data from individual cells, N = 10). Pseudo-colouring was generated by the ‘Fire’ lookup table in ImageJ. (D) Use-dependent activation was assessed under ambient…

• 

  Figure 5. Systematic mutagenesis of transmembrane cysteine residues in Kv1.2. (A) Cysteine residues are highlighted in red on the Kv1.2 structure (PDB 3LUT). C181, C229, C244, and C394 in the transmembrane domains were mutated to alanine. (B) Use-dependent activation of each cysteine mutant was assessed with trains of repetitive depolarizations, as described in Fig. 1, in ambient redox and after incubation in 666 µM DTT (N = 10–22 for each condition). (C) Schematic model depicting redox dependent interactions of Kv1.2 with a postulated extrinsic binding partner (blue) that mediates use-dependent activation. We propose that the reduced state of the regulatory partner has a high affinity for the Kv1.2 channel that promotes an inhibited gating mode by stabilizing the resting conformation. In more oxidizing conditions, the regulatory partner has weakened or altered interactions with Kv1.2, causing the inhibited gating mode to be less prominent.

• 

  Figure 6. Calibrated effects of redox potential on Kv1.2 use-dependent activation. (A,B) Extracellular redox potential was clamped using membrane impermeant redox couples ((A), cysteine and cystine (Cys:CySS), or (B), reduced and oxidized glutathione (GSH:GSSG)) by varying the ratio of the two species while maintaining a total concentration of 100 µM. N = 13–15 for each redox potential.