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 EC50 of 0.54 ± 0.068 mM and a predicted maximum shift of -38 mV. NFA at 1 mM also reduced maximum HCN2 conductance by ∼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.
Footnotes
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This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grant R01-HL65299].
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ABBREVIATIONS: NFA, niflumic acid (2-{[3-(trifluoromethyl)phenyl]amino}pyridine-3-carboxylic acid); WT, wild-type; G-V, conductance-voltage; HCN2, hyperpolarization-activated cyclic-nucleotide gated type 2; HCN2ntk, N-terminal truncated HCN2; I-V, current-voltage; τf, fast time constant for current activation; τs, slow time constant for current activation; τdeact, time constant for current deactivation; Vh, holding potential; Vt, test potential; V½, half-point of activation curve; UL-FS49, zatebradine; ZD7288, 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium chloride.
- Received December 23, 2008.
- Accepted February 13, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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