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 (EC 50 = 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 liad 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 (ECBO = 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+]c was elevated from 1 to 300 mM (EC30=21.2 mM). The slope conductance measured at the reversal potential exhibited the same [K+]e dependency (EC 50= 23.5 mM). Conductance was also [Na+]c 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+] c. Collectively, these findings indicate that Slo2.1 channel gating is modulated by [K-]c and [Na-]e., and that NFA uncouples channel activation from its modulation by transmembrane voltage and intracellular Na+. © 2010 Dai et al.