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Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid.

ABSTRACT: Niflumic acid (NFA) is a member of the fenamate class of nonsteroidal anti-inflammatory drugs. This compound and its derivatives are used worldwide clinically for the relief of chronic and acute pain. NFA is also a commonly used blocker of voltage-gated chloride channels. Here we present evidence that NFA is an efficient blocker of chloride-permeable glycine receptors (GlyRs) with subunit heterogeneity of action. Using the whole-cell configuration of patch-clamp recordings and molecular modeling, we analyzed the action of NFA on homomeric ?1?Ins, ?2B, ?3L, and heteromeric ?1? and ?2? GlyRs expressed in CHO cells. NFA inhibited glycine-induced currents in a voltage-dependent manner and its blocking potency in ?2 and ?3 GlyRs was higher than that in ?1 GlyR. The Woodhull analysis suggests that NFA blocks ?1 and ?2 GlyRs at the fractional electrical distances of 0.16 and 0.65 from the external membrane surface, respectively. Thus, NFA binding site in ?1 GlyR is closer to the external part of the membrane, while in ?2 GlyR it is significantly deeper in the pore. Mutation G254A at the cytoplasmic part of the ?1 GlyR pore-lining TM2 helix (level 2') increased the NFA blocking potency, while incorporation of the ? subunit did not have a significant effect. The Hill plot analysis suggests that ?1 and ?2 GlyRs are preferably blocked by two and one NFA molecules, respectively. Molecular modeling using Monte Carlo energy minimizations provides the structural rationale for the experimental data and proposes more than one interaction site along the pore where NFA can suppress the ion permeation.

SUBMITTER: Maleeva G

PROVIDER: S-EPMC5432571 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid.

Maleeva Galyna G Peiretti Franck F Zhorov Boris S BS Bregestovski Piotr P

Frontiers in molecular neuroscience 20170516

Niflumic acid (NFA) is a member of the fenamate class of nonsteroidal anti-inflammatory drugs. This compound and its derivatives are used worldwide clinically for the relief of chronic and acute pain. NFA is also a commonly used blocker of voltage-gated chloride channels. Here we present evidence that NFA is an efficient blocker of chloride-permeable glycine receptors (GlyRs) with subunit heterogeneity of action. Using the whole-cell configuration of patch-clamp recordings and molecular modeling ...[more]

PMID: 28559795

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Project description: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(+).

Dataset Information

Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid.

Publications

Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid.

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