K(V)4.3 N-terminal deletion mutant Δ2-39: effects on inactivation and recovery characteristics in both the absence and presence of KChIP2b

Abstract

Gating transitions in the K(V)4.3 N-terminal deletion mutant Δ2-39 were characterized in the absence and presence of KChIP2b. We particularly focused on gating characteristics of macroscopic (open state) versus closed state inactivation (CSI) and recovery. In the absence of KChIP2b Δ2-39 did not significantly alter the steady-state activation "a(4)" relationship or general CSI characteristics, but it did slow the kinetics of deactivation, macroscopic inactivation, and macroscopic recovery. Recovery kinetics (for both WT K(V)4.3 and Δ2-39) were complicated and displayed sigmoidicity, a process which was enhanced by Δ2-39. Deletion of the proximal N-terminal domain therefore appeared to specifically slow mechanisms involved in regulating gating transitions occurring after the channel open state(s) had been reached. In the presence of KChIP2b Δ2-39 recovery kinetics (from both macroscopic and CSI) were accelerated, with an apparent reduction in initial sigmoidicity. Hyperpolarizing shifts in both "a(4)" and isochronal inactivation "i" were also produced. KChIP2b-mediated remodeling of K(V)4.3 gating transitions was therefore not obligatorily dependent upon an intact N-terminus. To account for these effects we propose that KChIP2 regulatory domains exist in K(V)4.3 a subunit regions outside of the proximal N-terminal. In addition to regulating macroscopic inactivation, we also propose that the K(V)4.3 N-terminus may act as a novel regulator of deactivation-recovery coupling.

Figure 1

(A) Representative recordings of WT and “reduced amplitude” Δ2–39 currents elicited (HP = −100 mV) during a 2-s pulse to +50 mV, basal conditions. Peak transient current (peak current minus residual current at t = 2 s) amplitudes normalized (original peak current amplitudes: WT, 2121 nA; Δ2–39, 1428 nA). Calibration bar: 200 ms. Inset: Same currents on an expanded time scale. Dashed line, 50% peak current amplitude. Calibration bar: 100 ms. (B, C) Mean steady state activation “a⁴” and isochronal (1 sec) inactivation “i” relationships of WT (solid circles) and Δ2–39 (hollow circles) under (B) basal conditions (WT n = 20, Δ2–39 n = 17) and (C) + KChIP2b (“a⁴”: WT n = 15, Δ2–39 n = 24; “i”: WT n= 6, Δ2–39 n = 8). Mean “a⁴” data points fit to single Boltzmann relationships raised to the fourth power, mean “i” data points fit to single Boltzmann relationships. Fitting parameters summarized in Table 1. Solid gray curves in C) are corresponding fits of WT and Δ2–39 obtained under basal conditions as per (B). (D) Deactivation kinetics. Main panel: Mean (basal: WT n = 19, Δ2–39 n = 7; + KChIP2b: WT n = 13; Δ2–39 n= 11) τ_deact − V_m curves over the potential range −120 to −70 mV. Mean data points obtained from single exponential fits of deactivating tail currents. Basal black, + KChIP2b gray. Solid curves are single exponential fits to mean τ_deact data points [basal: WT e-fold change per 21.7 mV, Δ2–39 (fit from −120 to −80 mV) e-fold change per 15.0 mV; + KChIP2b: WT e-fold change per 33.8 mV, Δ2–39 (fit from −120 to −80 mV) e-fold change per 16.6 mV">basal: WT e-fold change per 21.7 mV, Δ2–39 (fit from −120 to −80 mV) e-fold change per 15.0 mV; + KChIP2b: WT e-fold change per 33.8 mV, Δ2–39 (fit from −120 to −80 mV) e-fold change per 16.6 mV]. Inset, Representative overlays of WT and Δ2–39 deactivating tail currents at −60 mV and −120 mV, basal conditions. Isochronal peak currents normalized for comparison and fit with single exponentials with following τ_deact values: −60 mV: WT, 24 ms; Δ2–39, 46 ms; α120 mV, WT, 4.4 ms; Δ2–39, 7.1 msec. Calibration bar: 10 ms.

Figure 2

Macroscopic inactivation kinetics at +50 mV (total 2 s pulse from combined and identical 1 second P1 and P2 pulses to +50 mV). Currents gray, fits black. (A) WT, basal conditions, representative double exponential fit with τ_fast = 67 ms, τ_slow = 328 ms, and A_fast = 0.817. Calibration bar: 500 nA, 200 ms. (B) Δ2–39, basal conditions, representative single exponential fit with τ = 179 ms that failed to acceptably converge to a double exponential fit (double exponential fit parameters [not illustrated]: τ_fast = 180.4 ms, τ_slow = 1327 ms, A_fast = 0.999). Calibration bar: 200 nA, 200 ms. (C) Δ2–39, basal conditions, representative comparison of single exponential (upper trace; τ = 238 ms) and double exponential (lower trace; τ_fast = 112 ms, τ_slow = 203 ms, and A_fast = 0.193) fits observed in the minority of cases (n = 7/22) where acceptable double exponential fits were also derived. Calibration bar: 500 nA, 200 ms. (D) Comparative net macroscopic inactivation kinetics at +50 mV. Representative overlays of normalized peak currents for WT, WT + KChIP2b, Δ2–39, and Δ2–39 + KChIP2b. Dashed line, 50% normalized peak current amplitude. Calibration bar: 200 ms.

Figure 3

Kinetics of recovery from macroscopic inactivation developed during a 1 second pulse to +50 mV, basal conditions. (A and B) Mean macroscopic recovery waveforms measured at HP = −100 mV to −70 mV for A) WT (n = 4–6) and B) Δ2–39 (n = 4–6). Dashed straight lines, 5o% relative P2 peak current amplitudes (see text). (C and D) Main panels: fits to WT (solid symbols) and Δ2–39 (hollow symbols) mean macroscopic recovery waveforms at C) HP = −100 mV and D) HP = −70 mV. C) HP = −100 mV: Black curves, sigmoidal fits using all mean data points. WT, “a²” fit, τ = 136 ms; Δ2–39, “a³” fit, τ = 127 ms. Blue curves, truncated single exponential fits beginning at initial times Δt. WT (Δt = 60 ms), τ = 178 ms; Δ2–39 (Δt = 100 ms), τ = 188 ms. D) HP = −70 mV: WT, black curve, single exponential fit using all data points, τ = 1,075 ms; Δ2–39, sigmoidal “a²” fit, τ = 990 ms. Blue curve, Δ2–39 truncated single exponential fit (Δt = 400 ms), τ = 1,733 ms. Insets: (C) Representative Δ2–39 macroscopic recovery waveform at HP = −100 mV. Black curve, sigmoidal “a³” fit, τ = 114 ms. Blue curve, single exponential fit (Δt = 120 ms), τ = 163 ms. Calibration bars: 1 µA, 100 ms. D) Potential dependence of mean times t_50% for half macroscopic recovery of WT (solid circles) and Δ2–39 (hollow circles). Extrapolated t_50% values determined by linear regression between the two immediate data points with mean values “above” and “below” 0.5 as per (A and B). Solid curves, single exponential fits with e-fold change in t_50% per mV as follows: WT, 15.0 mV; Δ2–39, 12.5 mV.

Figure 4

Effects of KChIP2b on macroscopic recovery kinetics (HP = −100 mV; protocol as per Fig. 3) of WT (solid black circles; n = 12) and Δ2–39 (hollow black circles; n = 13). Fits: WT + KChIP2b, single exponential with τ = 86 ms; Δ2–39 + KChIP2b: black sigmoidal curve, empirical “a^1.6” fit with τ = 108 ms; blue curve, truncated single exponential beginning at Δt = 60 ms with τ = 126 ms. Gray symbols and fits correspond to WT (solid circles) and Δ2–39 (hollow circles) obtained in the absence of KChIP2b (previously illustrated in Fig. 4C). Dashed line, t_50%. Extrapolated mean t_50% values (determined as described in Fig. 3 caption) as follows: WT + KChIP2b, 74 ms; Δ2–39 + KChIP2b, 116 ms; WT, 145 ms; Δ2–39, 200 ms. Inset: Δ2–39 + KChIP2b, representative macroscopic recovery waveform at HP = −100 mV. For the oocyte illustrated, black curve is an empirical sigmoid “a²” fit with τ = 76 ms, while blue curve is a single exponential fit (begun at Δt = 40 ms) with τ = 90 ms. Calibration bar: 300 nA, 100 ms.

Figure 5

(A) Kinetics and potential dependence of development of CSI. (A) Basal conditions. Main panel: Mean time constants τ_csi (derived from single exponential fits; see Inset) for development of CSI over the potential range of −70 to −40 mV. WT solid circles (n = 7–8), Δ2–39 hollow circles (n = 8–14). Combined aggregate mean data points from −60 to −40 mV fit with a single exponential function (solid black line; e-fold change in τ_csi per 7.3 mV). Inset: Δ2–39, representative exponential fit (τ = 1,702 ms) to development of CSI at −60 mV. Calibration bar: 200 nA, 500 ms. (B) + KChIP2b. Representative current recordings obtained during application of CSI development protocol at −70 mV of WT + KChIP2b (no to minimal development of CSI; no fit; calibration bars: 750 nA, 500 ms) and Δ2–39 + KChIP2b (significant development of CSI fit with single exponential with τ = 944 ms; calibration bars: 1 µA, 500 ms). (C) Mean kinetics of CSI at HP = −70 to −40 mV for WT + KChIP2b (measured only at −50 and −40 mV; solid black circles; n = 3) and Δ2–39 + KChIP2b (n = 5–8; hollow black circles). Mean Δ2–39 + KChIP2b data points fit (black curve) with a single exponential function (e-fold change per 10.33 mV). The gray data points and curve are mean WT (solid circles) and Δ2–39 (open circles) data obtained under basal conditions (as illustrated in A).

Figure 6

Recovery from CSI, basal conditions. (A) Representative recordings of recovery from CSI at HP = −100 mV for WT and Δ2–39. Empirical sigmoid “a²” fits: WT, τ = 195 ms; Δ2–39, τ = 203 ms. Calibration bars: WT 300 nA, 200 ms; Δ2–39 200 nA, 200 ms. (B) Mean CSI recovery waveforms for WT (solid symbols; n = 5–9) and Δ2–39 (hollow symbols; n = 5–9) at HP = −120 mV (diamonds), HP = −100 mV (circles), and HP = −80 mV (triangles). Similar mean CSI recovery curves were also measured for HP = −110 mV and HP = −90 mV [data not illustrated; these curves were used for extrapolation of t_50% values at HP = −110 and HP = −90 mV illustrated in (C)]. Dashed line, 50% relative P2 current amplitude. (C) Extrapolated mean t_50% values for recovery from CSI. Combined mean data points fit with a single exponential function (e-fold change per 12.4 mV).

Figure 7

Effects of KChIP2b on recovery from CSI. (A) Mean recovery waveforms from CSI at HP = −120, −100, and −80 mV (WT + KChIP2b, solid symbols, n = 4–7; Δ2–39 + KChIP2b, hollow symbols, n = 4–9). Relative peak current amplitudes normalized. Additional mean CSI recovery waveforms were measured at HP = −110 and −90 mV [mean data not illustrated; but see (B)]. Dashed line, 50% relative P2 current amplitude. (B) Δ2–39 + KChIP2b, representative CSI recovery waveforms measured at HP = −110 mV (upper panel: fit to waveform approximated with a single exponential with τ = 67 ms; calibration bars: 500 nA, 100 ms) and HP = −90 mV (lower panel: fit to waveform approximated with a single exponential with τ = 195 ms; calibration bars: 500 nA, 100 ms). Single exponential fits, while well describing the majority of peak currents measured at later times, nonetheless failed to adequately describe the earliest peak currents. (C) Mean extrapolated t_50% values for recovery from CSI. Mean data points fit with single exponential functions (WT + KChIP2b, e-fold change per 7.15 mV; Δ2–39 + KChIP2b, e-fold change per 9.78 mV). The gray curve is fit to WT and Δ2–39 t_50% values (as illustrated in Fig. 6C).