Activation of Slo2.1 channels by niflumic acid.
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 (EC(50) = 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 V(1/2) of +95 mV and an effective valence, z, of 0.48 e. Higher concentrations of NFA shifted V(1/2) to more negative potentials (EC(50) = 2.1 mM) and increased the minimum value of G/G(max) (EC(50) = 2.4 mM); at 6 mM NFA, Slo2.1 channel activation was voltage independent. In contrast, V(1/2) of the G-V relationship was shifted to more positive potentials when [K(+)](e) was elevated from 1 to 300 mM (EC(50) = 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(+)](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(+).
Dai
Li
L
Department of Physiology, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, UT 84112, USA.
Garg
Vivek
V
Sanguinetti
Michael C
MC
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015640:Ion Channel Gating; D008564:Membrane Potentials / Q000187:drug effects*; D009544:Niflumic Acid / Q000494:pharmacology*; D009865:Oocytes; D015221:Potassium Channels / Q000187:drug effects* / Q000378:metabolism; D000081033:Potassium Channels, Sodium-Activated; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism; D013329:Structure-Activity Relationship; D014981:Xenopus
D000894:Anti-Inflammatory Agents, Non-Steroidal; C480494:KCNT2 protein, human; D015221:Potassium Channels; D000081033:Potassium Channels, Sodium-Activated; D009544:Niflumic Acid; D012964:Sodium