A1899, PK-THPP, ML365, and Doxapram inhibit endogenous TASK channels and excite calcium signaling in carotid body type-1 cells

Abstract

Sensing of hypoxia and acidosis in arterial chemoreceptors is thought to be mediated through the inhibition of TASK and possibly other (e.g., BK_Ca ) potassium channels which leads to membrane depolarization, voltage-gated Ca-entry, and neurosecretion. Here, we investigate the effects of pharmacological inhibitors on TASK channel activity and [Ca²⁺ ]_i -signaling in isolated neonatal rat type-1 cells. PK-THPP inhibited TASK channel activity in cell attached patches by up to 90% (at 400 nmol/L). A1899 inhibited TASK channel activity by 35% at 400 nmol/L. PK-THPP, A1899 and Ml 365 all evoked a rapid increase in type-1 cell [Ca²⁺ ]_i . These [Ca²⁺ ]_i responses were abolished in Ca²⁺ -free solution and greatly attenuated by Ni²⁺ (2 mM) suggesting that depolarization and voltage-gated Ca²⁺ -entry mediated the rise in [Ca²⁺ ]_i. Doxapram (50 μmol/L), a respiratory stimulant, also inhibited type-1 cell TASK channel activity and increased [Ca²⁺ ]_i. . We also tested the effects of combined inhibition of BK_Ca and TASK channels. TEA (5 mmol/L) slightly increased [Ca²⁺ ]_i in the presence of PK-THPP and A1899. Paxilline (300 nM) and iberiotoxin (50 nmol/L) also slightly increased [Ca²⁺ ]_i in the presence of A1899 but not in the presence of PK-THPP. In general [Ca²⁺ ]_i responses to TASK inhibitors, alone or in combination with BK_Ca inhibitors, were smaller than the [Ca²⁺ ]_i responses evoked by hypoxia. These data confirm that TASK channel inhibition is capable of evoking membrane depolarization and robust voltage-gated Ca²⁺ -entry but suggest that this, even with concomitant inhibition of BK_Ca channels, may be insufficient to account fully for the [Ca²⁺ ]_i -response to hypoxia.

Keywords: TASK; Carotid body; Hypoxia; Kcnk3; Kcnk9; Potassium Channels.

Figure 1

(A) Representative recording of channel activity of TASK‐3, expressed in a HEK 293 cell, over ~2 min in a cell attached patch recording. Note marked inhibition of channel activity by 400 nmol/L PK‐THPP. (B) Data from the same recording over ~0.3 sec under control conditions (i) and in the presence of 400 nmol/L PK‐THPP (ii). (C) Bar chart comparing effects of 400 nmol/L PK‐THPP on single‐channel activity (nPopen) relative to control (n = 3, P < 0.001). Values are means ± SEM.

Figure 2

(A) Representative cell‐attached patch recording of channel activity for TASK‐1, expressed in a HEK 293 cell, over a ~3 min period demonstrating inhibition by 400 nmol/L A1899. (B) Data from same recording over ~0.1 sec intervals under control conditions (i) and in the presence of 400 nmol/L A1899 (ii). (C) Bar chart comparing effects of 400 nmol/L A1899 on single‐channel activity (nPopen) relative to control (n = 4, P = 0.045). Values are means ± SEM.

Figure 3

(A) Representative recording of type‐1 cell TASK channel activity over ~1.5 min in a cell‐attached patch showing inhibition of channel activity by 400 nmol/L PK‐THPP. (B) Data from same recording over ~1 sec intervals under control conditions (i) and in the presence of PK‐THPP. (C) Concentration‐response relationship of effects of PK‐THPP on rat type‐1 cell TASK channel activity (nPopen values in the presence of PK‐THPP expressed as % inhibition of control nPopen values). Data are mean ± SEM; n = 5 for 40 nm and 120 nmol/L PK‐THPP and n = 10 for 400 nmol/L PK‐THPP. Data were fit to a single‐site ligand binding model with r ² 0.997, B_{M ax} 100 and Kd 66 ± 10 nmol/L. (D) Representative all‐points frequency histogram showing profound reduction in current at all levels corresponding to channel open states. Each bar represents a 0.1 pA bin width. Data from analysis of 20 sec segments of cell‐attached recordings.

Figure 4

(A) Representative recording of type‐1 cell TASK channel activity over ~1.5 min of a cell‐attached patch recording demonstrating inhibition of channel activity by 400 nmol/L A1899. (B) Data from same recording over ~0.5 sec intervals under control conditions (i) and in the presence of A1899 (ii). (C) Concentration‐response relationship of effects of A1899 on type‐1 cell TASK channel activity (expressed as % inhibition in the presence of A1899 compared to control). Data are mean ± SEM (n = 4, 8 and 6 for 40, 400 and 4000 nmol/L concentrations of A1899, respectively). (D) Representative all‐points frequency histogram showing reduction of current at all levels corresponding to channel open states. Each bar represents a 0.1 pA bin width. Data were generated from analysis of 20 sec segments from cell‐attached recordings.

Figure 5

Effects of PK‐THPP on type‐1 cell [Ca²⁺]_i. (A) Recording showing effects of application of two different concentrations (120 and 400 nmol/L) of PK‐THPP causing an abrupt rise in [Ca²⁺]_i. (B) Concentration‐response graph of effects of PK‐THPP on [Ca²⁺]_i. Each point represents mean ± SEM (n = 5–7) and the curve is fit by non‐linear least squares regression to the equation Δ[Ca²⁺]_i = a  +  b. [PK‐THPP]/(EC50 +  [PK‐THPP]). The estimated EC50 is 45.2 nmol/L and the r ² for correlation is 0.19. (C) Effect of Ca²⁺ free Tyrode on [Ca²⁺]_i responses evoked by 400 nmol/L PK‐THPP. Note rapid reduction in [Ca²⁺]_i in the absence of external Ca²⁺. (D) Summary data showing effects of PK‐THPP on Δ[Ca²⁺]_i ([Ca²⁺]_i in the presence of PK‐THPP minus baseline [Ca²⁺]_i) under normal conditions and in the absence of extracellular Ca²⁺. Data are mean ± SEM. Statistical comparison is a paired t test. (E) Effect of 2 mmol/L Ni²⁺, a voltage‐gated Ca²⁺‐channel inhibitor, on [Ca²⁺]_i responses evoked by 400 nmol/L PK‐THPP. Note rapid reduction in [Ca²⁺]_i upon application of Ni²⁺. (F) Summary data showing effects of PK‐THPP on Δ[Ca²⁺]_i under normal conditions and in the presence of Ni²⁺. Data are mean ± SEM. Statistical comparison is a paired t test.

Figure 6

Effects of A1899 on type‐1 cell [Ca²⁺]_i. (A) Effects of application of three different concentrations (120, 400 and 1200 nmol/L) of A1899 demonstrating an abrupt, spiking, rise in [Ca²⁺]_i. (B) Concentration‐response graph of effects of A1899 on [Ca²⁺]_i. Each point represents mean ± SEM. The curve is fit by non‐linear least squares regression to the equation Δ[Ca²⁺]_i = a + b. [A1899]/(EC50 +  [A1899]). The estimated EC50 is 40.8 nmol/L and the r ² for correlation is 0.23. (C) Effect of Ca²⁺ free Tyrode on [Ca²⁺]_i responses evoked by 400 nmol/L A1899. Note absence of any [Ca²⁺]_i response to A1899 when applied in Ca²⁺ free Tyrode. (D) Summary data showing effects of A1899 on Δ[Ca²⁺]_i ([Ca²⁺]_i in the presence of A1899 minus baseline [Ca²⁺]_i) under normal conditions and in the absence of extracellular Ca²⁺.Statistical comparison is a paired t test. (E) Effect of 2 mmol/L Ni²⁺, a voltage‐gated Ca²⁺‐channel inhibitor, on [Ca²⁺]_i responses evoked by 400 nmol/L A1899. Note much smaller and slower rise in [Ca²⁺]_i when A1899 is applied in the presence of Ni²⁺. (F) Summary data showing effects of A1899 on Δ[Ca²⁺]_i under normal conditions and in the presence of Ni²⁺. Data are mean ± SEM. Statistical comparison is a paired t test.

Figure 7

Effects of ML365 on type‐1 cell [Ca²⁺]_i. (A) Representative trace showing effects of application of four different concentrations (4, 40, 400, and 4000 nmol/L) of ML365 demonstrating an abrupt rise in [Ca²⁺]_I in response to this TASK‐1 channel inhibitor. (B) Concentration‐response graph of effects of A1899 on [Ca²⁺]_i. Each point represents mean ± SEM (n = 8).

Figure 8

(A) Representative trace of Type‐1 cell TASK channel activity in a cell attached patch over ~1.5 min of recording, demonstrating reduction in activity by Doxapram. (B) Data from same recording over ~0.4 sec intervals under control conditions (i) and in the presence of doxapram (ii). (C) Concentration‐response relationship of doxapram on Type‐1 cell TASK channel activity. nPopen is plotted as % of that observed under control conditions. Doxapram was applied at 5 μmol/L (n = 3) and 50 μmol/L (n = 7). (D) Representative all‐points frequency histogram showing doxapram decreased all current levels corresponding to open channel activity (each bar represents a 0.1 pA bin width, data generated from 20 sec segments of cell‐attached recordings).

Figure 9

(A) Representative trace of the effect doxapram on glomus cell [Ca²⁺]i. (B) Summary of quantitative effect of doxapram on glomus cell [Ca²⁺]i. data are mean ± SEM n = 8, P < 0.001 (doxapram vs. control, paired t test).

Figure 10

Effects of 5 mmol/L TEA on the [Ca²⁺]_i response to TASK channel inhibitors. (A) TEA + 400 nmol/L PK‐THPP. (B) TEA + 400 nmol/L A1899. Note increase in size of [Ca²⁺]_i spikes in the presence of TEA.

Figure 11

Effects of 300 nmol/L paxilline on the [Ca²⁺]_i response to TASK channel inhibitors. (A) Paxilline + 400 nmol/L PK‐THPP. (B) Paxilline + 400 nmol/L A1899. Note increase in [Ca²⁺]_i response to A1899 during application of paxilline but that paxilline has no appreciable effects on the response to PK‐THPP.

Figure 12

Effects of 50 nmol/L Iberiotoxin on the [Ca²⁺]_i response to TASK channel inhibitors. (A) Iberiotoxin + 400 nmol/L PK‐THPP. (B) Iberiotoxin + 400 nmol/L A1899. Note apparent lack of effect of iberiotoxin in presence of PK‐THPP.

Figure 13

Summary of effects of combinations of K‐channel inhibitors on intracellular calcium. Y axis values represent the increase in [Ca²⁺]_i (over base line [Ca²⁺]_i) in the presence of K⁺‐channel inhibitor/s. [Ca²⁺]_i was averaged over the duration of exposure to the inhibitors (approx. 40–50 s). Experimental protocols are shown in Figures 8,9 and 10. Inhibitor concentrations were PK‐THPP 400 nmol/L, A1899 400 nmol/L, TEA 5 mmol/L, Paxilline (Pax) 300 nmol/L, and Iberiotoxin (Ibx) 50 nmol/L. Values are mean ± SEM, P values were calculated using a paired t test in all panels except (F) which uses a Wilcoxon signed‐rank test.

Figure 14

(A) Comparison of effects of K‐channel inhibitors with those of hypoxia (N₂ –equilibrated Tyrode see methods). Data (y axis) represents the average [Ca²⁺]_i response recorded in the presence of K⁺ channel inhibitor/s expressed as a percentage of the response to a hypoxic stimulus recorded in the same cell/s. The [Ca²⁺]_i response to inhibitor/hypoxia was determined from the average [Ca²⁺]_i recorded over an approx. 50 sec interval in the presence of inhibitor/hypoxia minus baseline [Ca²⁺]_i (recorded over 50 sec period under control conditions). Data are mean ± SEM. P values represent a comparison of response to inhibitor versus hypoxia using a paired t‐test. Note in all conditions bar one (which only narrowly failed to reach significance) the reponse to hypoxia was always greater than that to any single TASK‐channel inhibitor or combination of TASK and BK_{C a} channel inhibitor. Drug concentrations were PK‐THPP 400 nmol/L, A1899 400 nmol/L, Ml365 4 μmol/L, TEA 5 mmol/L, paxilline (Pax) 300 nmol/L, and iberiotoxin (Ibx) 50 nmol/L. (B) Box and whisker plot of all responses to hypoxia recorded in this study (n = 87). Box 25th‐75th percentiles, whisker 10th and 90th percentiles, broken line = mean, solid line = median. Individual outlying values only are plotted.

Figure 15

Effects of TASK channel inhibitors on calcium responses to high potassium and to hypoxia. (A) Representative trace of cellular calcium response to hypoxia alone, and in the presence of 400 nmol/L PK‐THPP. (B) Calcium response to depolarization by 30 mmol/L K⁺ alone, and in the presence of 400 nmol/L PK‐THPP. Increase in K⁺ was osmotically compensated for by reduction in Na⁺. (C) Calcium response to depolarization by 30 mmol/L K⁺ alone, and in the presence of 400 nmol/L A1899. (D) Summary of effects of TASK channel inhibitors on the calcium response to depolarization with 30 mmol/L K⁺ and the effects of PK‐THPP on the [Ca²⁺]_i response to hypoxia. Comparisons are i) Hypoxia versus Hypoxia + 400 nmol/L PK‐THPP, n = 6, P = 0.36, ii) 30 mmol/L K⁺ versus 30 mmol/L K⁺ + 400 nmol/L PK‐THPP, n = 7, P = 0.07, iii) 30 mmol/L K⁺ versus 30 mmol/L K⁺ + 400 nmol/L A1899, n = 6, P = 0.63.