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Niflumic Acid Alters Gating of HCN2 Pacemaker Channels by Interaction with the Outer Region of S4 Voltage Sensing Domains

Nora Eccles Harrison Cardiovascular Research and Training Institute and Department of Physiology, University of Utah, Salt Lake City, Utah

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.

Received for publication December 23, 2008.

Accepted for publication February 13, 2009.

Address correspondence to: Michael C. Sanguinetti, Nora Eccles Harrison Cardiovascular Research and Training Institute, Department of Physiology, University of Utah, 95 South 2000 East, Salt Lake City, UT 84112. E-mail: sanguinetti{at}cvrti.utah.edu