Activation of Slo2.1 channels by niflumic acid

Abstract

Slo2.1 channels conduct an outwardly rectifying K+ current when activated by high [Na+]i. Here, we show that gating of these channels can also be activated by fenamates such as niflumic acid (NFA), even in the absence of intracellular Na+. In Xenopus oocytes injected with <10 ng cRNA, heterologously expressed human Slo2.1 current was negligible, but rapidly activated by extracellular application of NFA (EC50 = 2.1 mM) or flufenamic acid (EC50 = 1.4 mM). Slo2.1 channels activated by 1 mM NFA exhibited weak voltage dependence. In high [K+]e, the conductance–voltage (G-V) relationship had a V1/2 of +95 mV and an effective valence, z, of 0.48 e. Higher concentrations of NFA shifted V1/2 to more negative potentials (EC50 = 2.1 mM) and increased the minimum value of G/Gmax (EC50 = 2.4 mM); at 6 mM NFA, Slo2.1 channel activation was voltage independent. In contrast, V1/2 of the G-V relationship was shifted to more positive potentials when [K+]e was elevated from 1 to 300 mM (EC50 = 21.2 mM). The slope conductance measured at the reversal potential exhibited the same [K+]e dependency (EC50 = 23.5 mM). Conductance was also [Na+]e dependent. Outward currents were reduced when Na+ was replaced with choline or mannitol, but unaffected by substitution with Rb+ or Li+. Neutralization of charged residues in the S1–S4 domains did not appreciably alter the voltage dependence of Slo2.1 activation. Thus, the weak voltage dependence of Slo2.1 channel activation is independent of charged residues in the S1–S4 segments. In contrast, mutation of R190 located in the adjacent S4–S5 linker to a neutral (Ala or Gln) or acidic (Glu) residue induced constitutive channel activity that was reduced by high [K+]e. Collectively, these findings indicate that Slo2.1 channel gating is modulated by [K+]e and [Na+]e, and that NFA uncouples channel activation from its modulation by transmembrane voltage and intracellular Na+.

Structure-Activity Relationship of Fenamates as Slo2.1 Channel Activators

“…As reported previously (Dai et al, 2010), negligible currents were observed in oocytes injected with low amounts of KCNT2 cRNA under control conditions (Fig. 1A, top).…”

“…KCNT2 cDNA (provided by L. Kaczmarek, Yale University, New Haven, CT) was subcloned into the psGEM oocyte expression vector (Dai et al, 2010). A278R Slo2.1 was generated by using the QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) and confirmed by DNA sequencing.…”

“…Whole-cell currents were recorded from oocytes using a standard two-microelectrode voltage-clamp technique (Stü hmer, 1992;Dai et al, 2010). Pipettes were pulled from borosilicate glass and tip-filled with 1% agarose dissolved in 3 M KCl and then back-filled with 3 M KCl to fabricate agarose-cushion microelectrodes (Schreibmayer et al, 1994).…”

“…The protein kinase C activator phorbol 12-myristate 13-acetate inhibits I Slo2.1 indirectly with an IC 50 value of 20 nM (Santi et al, 2006). We reported recently that the fenamates niflumic acid, 2-{[3-(trifluoromethyl)phenyl]amino}pyridine-3-carboxylic acid (NFA), and flufenamic acid, 2-[3-(trifluoromethyl)anilino]benzoic acid (FFA), can activate Slo2.1 with EC 50 values of 2.1 and 1.4 mM, respectively, in the absence of a change in [Na ϩ ] i (Dai et al, 2010). Although NFA is the first compound shown to activate Slo2.1, its low potency and nonspecificity severely limit its usefulness as a chemical probe in physiological studies.…”

“…As described for kidney CLC-K chloride channels (Liantonio et al, 2006), the biphasic effects of NFA on Slo2.1 could result from drug binding to distinct activator and inhibitory sites on the channel. Activation of Slo2.1 channels by extracellular NFA in inside-out patches (Dai et al, 2010) and the more accessible potent activity of external NFA on Slo1 channels expressed in lipid bilayers (Ottolia and Toro, 1994) indicate that the activator effect is mediated by interaction with an extracellular domain of the channel. The delayed and slow decay of current magnitude that follows the initial channel activation could result from the time required for the acidic drug to cross the cell membrane and inhibit channels by a second mechanism, either causing a direct pore block or a consequence of inhibition of cytosolic COX activity (e.g., inhibition by elevated levels of arachidonic acid).…”