Structure-activity relationship of fenamates as Slo2.1 channel activators

Abstract

Niflumic acid, 2-{[3-(trifluoromethyl)phenyl]amino}pyridine-3-carboxylic acid (NFA), a nonsteroidal anti-inflammatory drug that blocks cyclooxygenase (COX), was shown previously to activate [Na(+)](i)-regulated Slo2.1 channels. In this study, we report that other fenamates, including flufenamic acid, mefenamic acid, tolfenamic acid, meclofenamic acid, and a phenyl acetic acid derivative, diclofenac, also are low-potency (EC(50) = 80 μM to 2.1 mM), partial agonists of human Slo2.1 channels heterologously expressed in Xenopus oocytes. Substituent analysis determined that N-phenylanthranilic acid was the minimal pharmacophore for fenamate activation of Slo2.1 channels. The effects of fenamates were biphasic, with an initial rapid activation phase followed by a slow phase of current inhibition. Ibuprofen, a structurally dissimilar COX inhibitor, did not activate Slo2.1. Preincubation of oocytes with ibuprofen did not significantly alter the effects of NFA, suggesting that neither channel activation nor inhibition is associated with COX activity. A point mutation (A278R) in the pore-lining S6 segment of Slo2.1 increased the sensitivity to activation and reduced the inhibition induced by NFA. Together, our results suggest that fenamates bind to two sites on Slo2.1 channels: an extracellular accessible site to activate and a cytoplasmic accessible site in the pore to inhibit currents.

Fig. 1.

Effect of NFA on WT Slo2.1 channels. A, voltage-clamp protocol (top) and currents recorded from an oocyte expressing WT Slo2.1 channels before (middle, Control) and after treatment with 1 mM NFA (bottom). Oocytes were recorded after 3 days of injection with 0.4 ng of WT Slo2.1 cRNA. Arrows indicate 0 current. B, average I-V relationships for WT I_Slo2.1 recorded before (Control) and after treatment with 1 mM NFA (n = 4).

Fig. 2.

Biphasic action of NFA on Slo2.1. A, effects of 1 mM NFA on I_Slo2.1 recorded at a test pulse of 0 mV (left). a, current before the application of NFA. b, current at the peak of the activation response. c, current after 40 min of exposure to NFA. Time-dependent effect of 1 mM NFA on I_Slo2.1 (right). Currents were normalized to the peak activation response for each oocyte (n = 11). B, time-dependent activity of 1 mM NFA with the coapplication of the nonselective COX inhibitor ibuprofen (IBP, 1 mM) on I_Slo2.1 (left). a, current at 0 mV before the application of drugs. b, current at the peak of the activation response. c, current after 40 min of exposure to NFA + IBP. Time-dependent effect of NFA + IBP (blue square) and NFA alone (open circle) (right). Currents were normalized to the peak activation response for each oocyte. Average peak current for NFA treatment alone is 4.0 ± 0.4 μA (n = 6), and that for NFA + IBP is 4.9 ± 0.7 μA (n = 8). Data summarized in B were obtained from a single batch of oocytes.

Fig. 3.

A278R Slo2.1 channels are more sensitive to NFA. A, currents recorded from an oocyte expressing A278R mutant channels before (top, control) and after treatment with 1 mM NFA (bottom). Oocytes were recorded 1 day after the injection with 0.2 ng of cRNA. V_t was varied from −140 to +80 mV and applied in 20-mV increments from a holding potential of −80 mV. Arrows indicate 0 current. B, average I-V relationships for A278R I_Slo2.1 recorded before (Control) and after the treatment with 1 mM NFA (n = 10). C, NFA concentration-response relationships for WT (n = 9) and A278R (n = 13) I_Slo2.1 measured at 0 mV. Data were fitted with a logistic equation (smooth curve). For WT channels, EC₅₀ = 2.1 ± 0.1, n_H = 2.4 ± 0.06. For A278R channels, EC₅₀ = 0.11 ± 0.01, n_H = 2.0 ± 0.2. D, effects of 1 mM NFA on A278R I_Slo2.1 recorded at a test pulse of 0 mV. a, current recorded at a test pulse of 0 mV before the application of NFA. b, current at the peak of the activation response. c, current after 40 min of exposure to NFA. E, constitutively active A278R I_Slo2.1 is stable over 30 min of recording (Control, n = 7), whereas NFA-activated A278R I_Slo2.1 exhibits a 20% decline over 40 min (n = 11).

Fig. 4.

Concentration-response relationships for fenamates and diclofenac. A, chemical structures of fenamates and diclofenac. B and C, concentration-response relationships for the compounds on WT (B) and A278R (C) I_Slo2.1. For each compound, I_Slo2.1 was measured at 0 mV and normalized to the peak response. Data were fitted with a logistic equation (smooth curve) to determine the EC₅₀ values and n_H as presented in Table 1.

Fig. 5.

PAA is the minimal structural requirement for the activation of Slo2.1 channels. A, chemical structures of PAA (rings I and II are indicated), DPA, ANA, MAA, BCA, and BBA. B, average I-V relationship for WT I_Slo2.1 recorded before (Control) and after treatment with 3 mM PAA. C, traces of WT currents recorded at 0 mV before (Control) and after exposure of oocytes to the indicated concentrations of PAA. D, concentration-response relationship for PAA. EC₅₀ = 0.79 ± 0.03 mM, n_H = 1.5 ± 0.03 (n = 7). E–I, I-V relationships determined before (Control), after the treatment of oocytes with the indicated test compound, and then finally with 1 mM NFA (n = 3–5).

Fig. 6.

Apparent efficacies of several fenamates compared with that of NFA. An equieffective concentration (∼EC₉₀) of each fenamate was applied to oocytes expressing WT Slo2.1 (NFA, 5.4 mM; FFA, 2.9 mM; MFA, 1.65 mM; TFA, 0.98 mM; MCFA, 0.32 mM; PAA, 3.14 mM; DFS, 1.86 mM). The maximal response (I_peak at 0 mV) for each compound (n = 6–7) was normalized relative to the activation measured with NFA from the same batch of oocytes. The efficacies of MFA, MCFA, PAA, and DFS were less than that of NFA (*, p < 0.05; **, p < 0.01).

Fig. 7.

Time-dependent effects of PAA and MCFA on I_Slo2.1. A, effects of 1 mM PAA on I_Slo2.1 recorded at a test pulse of 0 mV (left). a, current before the application of PAA. b, peak current response. c, current after 40 min of PAA. Time-dependent effect of 1 mM PAA on I_Slo2.1 (right). Currents were normalized to the peak activation response for each oocyte (n = 10). B, effects of 0.03 mM MCFA on I_Slo2.1 recorded at a test pulse of 0 mV (left). a, current before the application of MCFA. b, peak current response. c, current after 40 min of MCFA. Time-dependent effect of 0.03 mM MCFA on I_Slo2.1 (right). Currents were normalized to the peak activation response for each oocyte (n = 9).

Fig. 8.

NFA activates Slo2.2 channels. A, currents recorded from an oocyte expressing Slo2.2 channels before (top) and after treatment with 1 mM NFA (bottom). Oocytes were recorded after 6 days of injection with 32 ng of WT Slo2.2 cRNA. Arrows indicate 0 current. B, average I-V relationships for WT I_Slo2.2 recorded before (Control) and after treatment with the indicated concentrations of NFA (n = 7). C, concentration-response relationships for NFA on I_Slo2.2 measured at 0 mV and normalized to the peak response (n = 7). Data were fitted with a logistic equation to determine the EC₅₀ value (2.7 ± 0.19 mM) and n_H (2.0 ± 0.14).

Fig. 9.

Effects of fenamates on Slo1 channels. A–C, currents recorded from oocytes expressing Slo1 channels before (top) and after treatment with 1 mM of the indicated fenamate (bottom). Arrows indicate 0 current. D–F, concentration-dependent effects of NFA (n = 5), MCFA (n = 11), and PAA (n = 7) on averaged I-V relationships. Oocytes were recorded after 1 to 3 days of injection with 0.04 to 1.0 ng of WT Slo1 cRNA. G, concentration-response relationships for the indicated fenamate. Data were fitted with a logistic equation (smooth curves) to estimate the EC₅₀ value. For MCFA, the EC₅₀ value was 0.68 ± 0.19 (n_H = 2.6 ± 0.4; n = 11). For NFA and PAA, n_H was fixed at 2.4 and 1.5, respectively, to estimate EC₅₀ values of 10.0 ± 0.9 mM (n = 5) for NFA and 13.4 ± 0.7 mM (n = 7) for PAA. For NFA, I_Slo1 was measured at 0 mV, and responses in uninjected oocytes were used to correct for the activation of endogenous currents. For MCFA and PAA, I_Slo1 was measured at +60 mV without correction, because the endogenous currents evoked by these compounds were very small. H, apparent efficacy of Slo2.2 channel activation for MCFA compared with that for NFA. The maximal response (I_peak at 0 mV) for each compound (n = 5–11) was normalized relative to the activation measured with NFA from the same batch of oocytes. The efficacy of MCFA was less than that of NFA (*, p < 0.001).