Niflumic acid alters gating of HCN2 pacemaker channels by interaction with the outer region of S4 voltage sensing domains

Abstract

Niflumic acid, 2-[[3-(trifluoromethyl)phenyl]amino]pyridine-3-carboxylic acid (NFA), is a nonsteroidal anti-inflammatory drug that also blocks or modifies the gating of many ion channels. Here, we investigated the effects of NFA on hyperpolarization-activated cyclic nucleotide-gated cation (HCN) pacemaker channels expressed in X. laevis oocytes using site-directed mutagenesis and the two-electrode voltage-clamp technique. Extracellular NFA acted rapidly and caused a slowing of activation and deactivation and a hyperpolarizing shift in the voltage dependence of HCN2 channel activation (-24.5 +/- 1.2 mV at 1 mM). Slowed channel gating and reduction of current magnitude was marked in oocytes treated with NFA, while clamped at 0 mV but minimal in oocytes clamped at -100 mV, indicating the drug preferentially interacts with channels in the closed state. NFA at 0.1 to 3 mM shifted the half-point for channel activation in a concentration-dependent manner, with an EC(50) of 0.54 +/- 0.068 mM and a predicted maximum shift of -38 mV. NFA at 1 mM also reduced maximum HCN2 conductance by approximately 20%, presumably by direct block of the pore. The rapid onset and state-dependence of NFA-induced changes in channel gating suggests an interaction with the extracellular region of the S4 transmembrane helix, the primary voltage-sensing domain of HCN2. Neutralization (by mutation to Gln) of any three of the outer four basic charged residues in S4, but not single mutations, abrogated the NFA-induced shift in channel activation. We conclude that NFA alters HCN2 gating by interacting with the extracellular end of the S4 voltage sensor domains.

Fig. 1.

NFA slows the rate of onset of WT HCN2 current activation and shifts its voltage dependence of activation to more negative potentials. A and B, WT HCN2 channel currents recorded from a single oocyte during 5-s pulses to test potentials ranging from -10 to -130 mV, applied in 10-mV steps. The V_h was -30 mV, recorded at a V_t of -90 mV (C) and -120 mV (D). Traces recorded before (control) and after exposure to 1 mM NFA are superimposed. At -90 mV, control: τ_f = 489 ms, τ_s = 2400 ms; A_f/(A_f + A_s) = 0.64; 1 mM NFA: τ = 3239 ms. At -120 mV, control: τ_f = 140 ms, τs = 448 ms; A_f/(A_f + A_s) = 0.96; 1 mM NFA: τ_f = 258 ms, τs = 1271 ms; A_f/(A_f + A_s) = 0.65. E, I-V relationship measured before and after NFA. Currents were measured at the end of the 5-s pulse to the indicated V_t. F, effect of NFA on the voltage dependence of current activation. Currents were not corrected for instantaneous leak. Under control conditions the V_½ was -69 mV (k = 7.1 mV); after 1 mM NFA, V_½ was -96 mV and k was 6.5 mV. All data are from the same oocyte.

Fig. 2.

State-dependent modification of WT HCN2 channels by NFA. A, test for effects of NFA on closed channels. Top, voltage pulse protocol and timing of NFA exposure. A test pulse was applied before NFA, and once again after 3 min application of 1 mM NFA. The V_h was 0 mV between test pulses. Lower panel shows the two test currents on an expanded time scale. Current activation was elicited with a 2-s pulse to -100 mV followed by a 1-s pulse to +20 mV to induce deactivation. Current magnitude was defined as time-dependent inward current measured at the end of pulse (indicated by arrows) minus the instantaneous current at start of pulse. B, test for effects of NFA on open channels. Top, voltage pulse protocol. Bottom, inward current at a V_h of -100 mV, the times where current magnitude was measured (indicated by arrows) and the timing of NFA exposure. C, reduction of HCN2 current by NFA, defined as percentage of time-dependent current measured at -100 mV after 3 min of drug compared with current measured before drug exposure.

Fig. 3.

NFA slows the activation rate and shifts the voltage dependence of activation of WT HCN2_ntk channel currents to more negative potentials. A and B, WT HCN2_ntk channel currents recorded from a single oocyte during 5-s pulses to test potentials ranging from -10 to -130 mV, applied in 10-mV steps, before (A) and after application of 1 mM NFA(B). The V_h was -30 mV and a pulse to -130 mV was applied after each test pulse. C, NFA slowed the rate of activation and deactivation of HCN_ntk channel current. For control: τ_f = 159 ms, τ_s = 1315 ms; A_f/(A_f + A_s) = 0.87. τ_deact = 80 ms,162 ms, A_f/(A_f + A_s) = 0.84. For 1 mM NFA: τ_f = 366 ms, τ_s = 1944ms; A_f/(A_f + A_s) = 0.60. τ_deact = 145 ms,1110 ms, A_f/(A_f + A_s) = 0.62. D, I-V relationship measured before and after NFA. Currents were measured at the end of the 5-s pulse to the indicated V_t. E, voltage dependence of current activation Control: V_½ = -81 mV, k = 6.48 mV; 1 mM NFA: V_½ = -108 mV; k = 6.58 mV. All data are from the same oocyte.

Fig. 4.

NFA causes concentration-dependent shift in the voltage dependence of WT HCN_ntk channel activation. A, normalized I-V relationships recorded before and after treatment of oocytes to 1 mM NFA (n = 16). For most data points, the small S.E. bars are masked by the larger symbols. B, effect of 1 mM NFA on normalized voltage dependence of current activation (G-V relationship). The V_½ of WT HCN_ntk channels was -77.6 ± 0.9 mV and slope factor, k was 7.5 ± 0.2 mV; after treatment with 1 mM NFA, V_½ was -102.0 ± 1.2 mV and k was 7.7 ± 0.2 mV (n = 16). The dotted curve represents NFA data normalized to its own peak value. C, [NFA]-dependent shift in voltage dependence of HCN2_ntk channel activation. The EC₅₀ for the NFA-induced shift in V_½ was 542 ± 68 μM (n_H = 0.97). The maximum shift in V_½ predicted by extrapolation of the Hill plot was -38 mV. D, kinetics of activation. Plotted data represent the average fast (open symbols) and slow (filled symbols) time constants for the onset of current activation during 5-s pulses at the indicated V_t. ^*p < 0.01 compared with matched control.

Fig. 5.

Neutralization of single basic residues in S4 domain does not prevent NFA-induced negative shift in the voltage dependence of activation of HCN2_ntk channels. A, sequence of the HCN2 S4 domain. B to F, normalized I-V relationships recorded before and after treatment of oocytes to 1 mM NFA. The single mutation introduced into the Arg or Lys residue in the S4 domain is indicated for each channel type. Smooth curves represent Boltzmann fit to the data. The dotted curves represent NFA data normalized to its own peak value. The values for V_½ and k for the Boltzmann function are listed in Table 1.

Fig. 6.

Effect of 1 mM NFA on activation kinetics of HCN2_ntk channels containing a single S4 mutation. The S4 residue mutation is indicated at the top of each graph. Plotted data represent the average fast (open symbols) and slow (filled symbols) time constants for the onset of current activation during 5-s pulses at the indicated V_t. ^*p < 0.01; ^**, p < 0.05 compared with matched control.

Fig. 7.

Neutralization of four outer basic residues in S4 domain abolishes NFA-induced negative shift in HCN2_ntk channel activation. A, quadruple mutant channel currents recorded from an oocyte during 5-s activating pulses to a test potentials of -140 mV, followed by pulse to +20 mV to record channel deactivation. For control: τ_f = 67 ms, τ_s = 1017 ms; A_f/(A_f + A_s) = 0.83. τ_deact = 154 ms. For 1 mM NFA: τ_f = 98 ms, τ_s = 1008 ms; A_f/(A_f + A_s) = 0.81. τ_deact = 194 ms. B, effect of 1 mM NFA on the kinetics of activation. Plotted data represent the average fast (open symbols) and slow (filled symbols) time constants for the onset of current activation during 5-s pulses at the indicated V_t. ^*, p < 0.01; ^**, p < 0.05 compared with matched control. C and D, normalized G-V relationships recorded before and after treatment of oocytes to 1 mM NFA. The test pulse durations used to elicit current were either 1 s (C) or 5 s (D). Smooth curves represent Boltzmann fit to the data. The dotted curves represent NFA data normalized to its own peak value. The values for V_½ and k for the Boltzmann function are listed in Table 1.

Fig. 8.

Neutralization of outer three basic residues in S4 domain prevents NFA-induced negative shift in the voltage dependence of activation of HCN2_ntk channels. A, triple mutant (K291Q/R294Q/R297Q) channel currents recorded from an oocyte during 5-s activating pulse to -100 mV, followed by pulse to + 20 mV to record channel deactivation. For control: τ_f = 179 ms, τ_s = 1539 ms; A_f/(A_f + A_s) = 0.56. τ_deact = 162 ms. For 1 mM NFA: τ_f = 228 ms, τ_s = 1497 ms; A_f/(A_f + A_s) = 61. τ_deact = 217 ms. B, effect of 1 mM NFA on the kinetics of activation. Plotted data represent the average fast (open symbols) and slow (filled symbols) time constants for the onset of current activation during 5-s pulses at the indicated V_t. ^*, p < 0.01; ^**, p < 0.05 compared with matched control. C and D, normalized G-V relationships recorded before and after treatment of oocytes to 1 mM NFA. The test pulse durations used to elicit current were either 1 s (C) or 5 s (D). Smooth curves represent Boltzmann fit to the data. The dotted curves represent NFA data normalized to its own peak value.

Fig. 9.

Neutralization of Arg294, Arg297, and Arg300 residues in S4 domain prevents NFA-induced negative shift in the voltage dependence of activation of HCN2_ntk channels. A, triple mutant (R294Q/R297Q/R300Q) channel currents recorded from an oocyte during 5-s activating pulse to -130 mV, followed by pulse to + 20 mV to record channel deactivation. For control: τ_f = 179 ms, τ_s = 1056 ms; A_f/(A_f + A_s) = 0.70. τ_deact = 167 ms. For 1 mM NFA: τ_f = 188 ms, τ_s = 1332 ms; A_f/(A_f + A_s) = 0.71. τ_deact = 189 ms. B, effect of 1 mM NFA on the kinetics of activation. Plotted data represent the average fast (open symbols) and slow (filled symbols) time constants for the onset of current activation during 5-s pulses at the indicated V_t. ^*, p < 0.01; ^**, p < 0.05 compared with matched control. C and D, normalized G-V relationships recorded before and after treatment of oocytes to 1 mM NFA. The test pulse durations used to elicit current were either 1 s (C) or 5 s (D). Smooth curves represent Boltzmann fit to the data. The dotted curves represent NFA data normalized to its own peak value.

Fig. 10.

Summary of NFA-induced shifts in V_½ for activation of HCN_ntk channel current. Voltage dependence of activation was determined using 5-s activating pulses before and after treatment of oocytes with 1 mM NFA. Number of oocytes is indicated at the end of each bar.