Inhibition of hKv2.1, a major human neuronal voltage-gated K+ channel, by meclofenamic acid
Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs), such as fenamate, aspirin, acetaminophen and ibuprofen, are widely used substances. The fenamate NSAIDs such as meclofenamic, flufenamic, mefenamic and niflumic acids are all derivatives of N-phenylanthranilic acid (Fig. 1). The potential effects of NSAIDs on the central nervous system, such as neuro-protection in Alzheimer's disease (Rich et al., 1995; Stewart et al., 1997), have spurred several basic and clinical studies. It is well known that the major mechanism mediating anti-inflammatory effects of fenamate and other NSAIDs is inhibition of the cyclooxygenase that catalyzes the biosynthesis of prostaglandins (e.g., prostaglandin E2) from arachidonic acid in both peripheral and central tissues (Wu, 1998). However, accumulating evidence indicates that fenamates also modulate a diversity of ion channels through a pathway which may be independent of the cyclooxygenase-prostaglandin mechanism. For example, it has been reported that meclofenamic acid (5–10 μM) is a potent blocker of an ATP-sensitive K+ channel (Grover et al., 1994). The effect of niflumic acid on ion channels seems more complex as it blocks both anionic and cationic channels in a number of species at various concentrations from 10–100 μM (White and Aylwin, 1990; Gögelein et al., 1990; Hogg et al., 1994; Wang et al., 1997a, Wang et al., 1997b). In addition, niflumic acid and other fenamates potentiate a large conductance Ca2+-activated K+ channel current, possibly by acting directly on the channel proteins at concentrations from 75 μM to 1 mM (Farrugia et al., 1993; Ottolia and Toro, 1994; Greenwood and Large, 1995). Effects of fenamates on ion channels in the central nervous system have also been reported. For example, fenamates modulate the GABAA receptor-gated Cl− channels in a concentration-dependent fashion. When Cl− currents were induced by low concentrations of GABA, fenamates potentiated this ligand-gated current with EC50s of 5–10 μM. In contrast, the maximum Cl− currents stimulated by a high GABA concentration (3 mM) were inhibited by fenamates with IC50s of 7–50 μM (Woodward et al., 1994).
Despite the above-mentioned reports, little is known about the effects of fenamates on neuronal voltage-gated K+ channels (Kv). It is well established that Kv channels are crucial in shaping action potentials (Hille, 1992; Pongs, 1992). Ultimately, Kv channels are involved in the regulation of intracellular calcium level and thereby neurotransmitter release (Boireau et al., 1991; Schechter, 1997). To date, at least five subfamilies of functional Kv channel α subunits have been cloned (Kv1-5) and their expression pattern in mammalian central nervous system has been extensively investigated (Pongs, 1992; Jan and Jan, 1997). Recent data from Wang et al. (1997a)have shown that some fenamates at high concentration (1 mM) may act as blockers of the Kv4 subfamily which encode “A-type” transient K currents. However, Kv4 subfamily exists not only in central nervous system, but also in the heart. The majority Kv channels in the soma are Kv2.1 and virtually every neuron in the brain expresses Kv2.1 whereas Kv1 subfamily is expressed in the axonal terminals (Awan and Dolly, 1991; Pongs, 1992; Trimmer, 1993; Rhodes et al., 1997; Rasband et al., 1998; Murakoshi and Trimmer, 1999). Physiological significance of these Kv channels is evident as behavioral changes in mice were observed when a Kv channel was “knocked-down” using an intracerebroventricular injection of antisense oligonucleotide (Galeotti et al., 1997) or knocked out genetically (Galeotti et al., 1997; Smart et al., 1998). Therefore, the primary goal of this investigation was to find out if fenamates were blockers of Kv2.1 K+ channels. For comparison, we also performed some experiments with another delayed rectifier K channel, the hKv1.1. Both channels were stably expressed in the Chinese hamster ovary (CHO) cells. The results demonstrate that meclofenamic acid preferentially inhibits hKv2.1 in a concentration-dependent manner. This inhibition seems independent of the cyclooxygenase-prostaglandin pathway.
Section snippets
Construction of CHOK1-hKv1.1 and CHOK1-hKv2.1 cell line
Methods for the construction of cell lines were similar to what had been reported previously (Wang et al., 1998). Human Kv1.1 cDNA (kind gift of Dr. Bruce Tempel) was excised from pGexHG2 with BglII/EcoRI and inserted into the BamHI/EcoR1 sites of mammalian expression vector pWE1 to yield pWE1/Kv1.1. The construct was characterized by restriction enzyme mapping and DNA sequencing (dideoxynucleotide chain termination method). CHOK1 cells were transfected with linearized pWE1/Kv1.1 by
Inhibition of Kv channel currents by fenamates
In order to produce a preliminary pharmacological profile of the four fenamates on Kv channels, we used a single dose (100 μM) of fenamates in our first set of experiments. Fig. 2A shows that meclofenamic acid (100 μM) inhibited hKv2.1 current by over 50%. Another fenamate NSAID, niflumic acid (100 μM), was almost without effect on hKv2.1 current (Fig. 2B). The effects of meclofenamic acid on Kv channels were readily reversible upon wash. In three cells at a test potential of +50 mV 100 μM
Discussion
The current study clearly showed that the NSAID meclofenamic acid inhibits hKv2.1 (a delayed rectifier K+ channel) current amplitude in a concentration-dependent manner with little effect on the channel activation and deactivation kinetics. The data also suggested that this inhibition is likely due to a direct interaction of the drug with the channel proteins.
NSAIDs including fenamates are known to block the cyclooxygenase-prostaglandin pathway to achieve their anti-inflammatory effects in both
Acknowledgements
The authors wish to thank Drs. James Barrett, Paul McGonigle and Kenneth Rhodes for reading our manuscript and providing crucial suggestions.
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