Apoptotic proteins Reaper and Grim induce stable inactivation in voltage-gated K+ channels

Abstract

Drosophila genes reaper, grim, and head-involution-defective (hid) induce apoptosis in several cellular contexts. N-terminal sequences of these proteins are highly conserved and are similar to N-terminal inactivation domains of voltage-gated potassium (K+) channels. Synthetic Reaper and Grim N terminus peptides induced fast inactivation of Shaker-type K+ channels when applied to the cytoplasmic side of the channel that was qualitatively similar to the inactivation produced by other K+ channel inactivation particles. Mutations that reduce the apoptotic activity of Reaper also reduced the synthetic peptide's ability to induce channel inactivation, indicating that K+ channel inactivation correlated with apoptotic activity. Coexpression of Reaper RNA or direct injection of full length Reaper protein caused near irreversible block of the K+ channels. These results suggest that Reaper and Grim may participate in initiating apoptosis by stably blocking K+ channels.

Figure 1

Short synthetic apoptosis peptides induce inactivation in voltage-dependent ShBΔ6–46:T449V K⁺ channels. (A) N-terminal sequences of the ShB K⁺ channel, Reaper, Grim, and Hid. The number of amino acids shown corresponds to the length of synthetic peptides used in the experiments. Peptides were synthesized as described in Materials and Methods. (B) Representative macroscopic currents recorded from an inside-out patch at +50 mV in the presence of the ShB, Reaper, Grim, or Hid peptide. Peptides were applied to the cytoplasmic side of the membrane patch at 100 μM concentration. (C) Concentration dependence of the fractional steady-state block of the ShBΔ6–46:T449V current by the Reaper and Grim N-terminal peptides (mean ± SD, n = 5). Steady-state currents were measured in response to pulses to +50 mV for 200 ms to 1.5 s in duration. Fraction of the steady-state current blocked is plotted as a function of the peptide concentration. (D) Effect of the Reaper N terminus peptide on openings of a single ShBΔ6–46:T449V channel. Representative openings at 0 mV with and without the Reaper peptide are shown. The upper three sweeps were recorded in the absence of and the lower four sweeps were recorded in the presence of Reaper peptide (100 μM). The open time histograms generated from the openings without peptide (upper) and with peptide (lower) also are shown. Solid lines in the histograms represent single exponential fits to the data. The mean open times for the control and peptide data were 12.8 ms and 4.4 ms, respectively. (E) Concentration dependence of the reciprocal of the mean open time. The reciprocals of the mean open times, measured in the presence of different concentrations of the Reaper N-terminal peptide, are plotted as a function of the peptide concentration. The different symbols show the results obtained from three different experiments. The straight line shows the concentration dependence as predicted from the open channel scheme discussed in the text. The slope of the line, which represents k_ON, was 1.6 s⁻¹ μM⁻¹ and the y intercept was 75 s⁻¹. (F) Rate constants (k_ON and k_OFF) of the block of ShBΔ6–46:T449V currents by the synthetic N-terminal peptides. Rate constants were estimated from macroscopic current recordings as described in Materials and Methods. The estimated values of blocking and unblocking rate constants, k_ON and k_OFF, respectively, are compared by using boxplots.

Figure 2

Mutations known to affect Reaper’s cell killing ability also affect K⁺ channel blocking ability. (A) Blocking effects of Reaper N terminus peptides (wild-type, F5A, and Y6A) on ShBΔ6–46:T449V currents. Currents recorded from an inside-out patch at +50 mV are shown. Peptides were applied at 100 μM. (B) Concentration dependence of the channel block by the Reaper wild-type, F5A, and Y6A N terminus peptides (mean ± SD, n = 5). Macroscopic ShBΔ6–46:T449V currents were measured in response to depolarization to +50 mV for 200 ms to 1.5 s in duration. Normalized steady-state current amplitudes are plotted as a function of the peptide concentration. (C) Rate constants (k_ON and k_OFF) of the block by the Reaper wild-type, F5A, and Y6A N terminus peptides. Rate constants were estimated by fitting the macroscopic current time course in the presence of the peptides at 100 μM as described in Materials and Methods. Values of the rate constants are compared by using boxplots. (D) Recovery from the Reaper F5A peptide-induced block is faster. Time course of recovery from peptide-induced block was estimated by using a standard double-pulse protocol. Two depolarizing pulses to +50 mV, separated by various intervals as indicated, were applied. Relative current amplitudes, amplitude elicited by the second pulse divided by that from the first pulse, are plotted. Representative ShBΔ6–46:T449V currents recorded in the presence of the Reaper F5A peptide during the second pulses to +50 mV are shown (left). Recovery time courses observed with wild-type and F5A Reaper peptides (100 μM) are compared (right; mean ± SD, n = 3). Smooth lines represent single exponential fits to the data. Recovery time constants for wild-type and F5A Reaper were 70 ms and 30 ms, respectively. (E) The A11N mutation does not affect the Reaper N terminus peptide’s ability to block the channel. Reaper wild-type and A11N peptides were applied at 100 μM. The time courses of the currents recorded in the presence of the two peptides were similar at all concentrations examined (1 to 100 μM, data not shown).

Figure 3

Effects of full length Reaper protein on the K⁺ channel. (A) Coinjection of Reaper and ShBΔ6–46:T449V RNAs into Xenopus laevis oocytes inhibits functional expression of the channel. Two-electrode voltage-clamp recordings at +50 mV were made 1 day after injection of the RNAs. Solid lines represent the averaged currents from 13 cells. Shaded areas represent the respective SEM. (B) Reaper RNA injection reduces K⁺ currents through the ShBΔ6–46:T449V channels already in the membrane. Oocytes were injected with ShBΔ6–46:T449V channel RNA on Day 0. On Day 1, the cells were divided into two groups, and control recordings were made (current amplitudes at 0 hr). Cells then were injected with either Reaper RNA or H₂O (control). Currents again were recorded 24 hr later (current amplitude values at 24 hr). Injection of Reaper RNA resulted in significant reduction in current amplitude (right) whereas injection of H₂O had no effect (left). Solid lines represent mean currents recorded by using a two-electrode voltage clamp from 10 to 15 cells. Shaded areas represent the respective SEM. (C) Direct injection of synthetic full length Reaper peptide decreases the ShBΔ6–46:T449V current amplitude. Control currents were recorded by using a two-electrode voltage-clamp, and the cells then were injected immediately with either H₂O (40 nl) or full length synthetic Reaper protein (40 nl, 1 mM). Cells were allowed to recover for 2 hr after injection, and currents then were recorded from the same cells. Representative currents before and after peptide injection are shown (left). Relative changes in the current amplitudes are compared by using boxplots (right). (D) Synthetic full length Reaper peptide induces inactivation in the ShBΔ6–46:T449V K⁺ channel. Macroscopic currents recorded from one patch are shown. The control current, the current recorded in response to the 18th pulse in the presence of Reaper, and the current after wash-out are shown. Depolarizing pulses to +50 mV were applied every 10 s. (E) The effect of synthetic full length Reaper is near irreversible. Peak macroscopic ShBΔ6–46:T449V current amplitudes recorded from one inside-out patch in response to repeated depolarizing pulses to +50 mV from a holding potential of −90 mV at 10-s intervals are shown. Synthetic Reaper N terminus peptide (100 μM) and full length Reaper synthetic peptide (100 μM) were applied to the intracellular side of the patch. The block induced by the short N terminus peptide (N-term) was completely reversible by washing the chamber with peptide-free solution. In contrast, channel block induced by full length Reaper peptide (Full) was only partially reversible. Hyperpolarization to −140 mV resulted in only a slight increase in peak current amplitude.