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Inhibition of human N- and T-type calcium channels by an ortho-phenoxyanilide derivative, MONIRO-1.

ABSTRACT: BACKGROUND AND PURPOSE:Voltage-gated calcium channels are involved in nociception in the CNS and in the periphery. N-type (Cav 2.2) and T-type (Cav 3.1, Cav 3.2 and Cav 3.3) voltage-gated calcium channels are particularly important in studying and treating pain and epilepsy. EXPERIMENTAL APPROACH:In this study, whole-cell patch clamp electrophysiology was used to assess the potency and mechanism of action of a novel ortho-phenoxylanilide derivative, MONIRO-1, against a panel of voltage-gated calcium channels including Cav 1.2, Cav 1.3, Cav 2.1, Cav 2.2, Cav 2.3, Cav 3.1, Cav 3.2 and Cav 3.3. KEY RESULTS:MONIRO-1 was 5- to 20-fold more potent at inhibiting human T-type calcium channels, hCav 3.1, hCav 3.2 and hCav 3.3 (IC50 : 3.3 ± 0.3, 1.7 ± 0.1 and 7.2 ± 0.3 ?M, respectively) than N-type calcium channel, hCav 2.2 (IC50 : 34.0 ± 3.6 ?M). It interacted with L-type calcium channels Cav 1.2 and Cav 1.3 with significantly lower potency (IC50 > 100 ?M) and did not inhibit hCav 2.1 or hCav 2.3 channels at concentrations as high as 100 ?M. State- and use-dependent inhibition of hCav 2.2 channels was observed, whereas stronger inhibition occurred at high stimulation frequencies for hCav 3.1 channels suggesting a different mode of action between these two channels. CONCLUSIONS AND IMPLICATIONS:Selectivity, potency, reversibility and multi-modal effects distinguish MONIRO-1 from other low MW inhibitors acting on Cav channels involved in pain and/or epilepsy pathways. High-frequency firing increased the affinity for MONIRO-1 for both hCav 2.2 and hCav 3.1 channels. Such Cav channel modulators have potential clinical use in the treatment of epilepsies, neuropathic pain and other nociceptive pathophysiologies. LINKED ARTICLES:This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

SUBMITTER: McArthur JR

PROVIDER: S-EPMC5980596 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Inhibition of human N- and T-type calcium channels by an ortho-phenoxyanilide derivative, MONIRO-1.

McArthur Jeffrey R JR Motin Leonid L Gleeson Ellen C EC Spiller Sandro S Lewis Richard J RJ Duggan Peter J PJ Tuck Kellie L KL Adams David J DJ

British journal of pharmacology 20170721 12

<h4>Background and purpose</h4>Voltage-gated calcium channels are involved in nociception in the CNS and in the periphery. N-type (Ca<sub>v</sub> 2.2) and T-type (Ca<sub>v</sub> 3.1, Ca<sub>v</sub> 3.2 and Ca<sub>v</sub> 3.3) voltage-gated calcium channels are particularly important in studying and treating pain and epilepsy.<h4>Experimental approach</h4>In this study, whole-cell patch clamp electrophysiology was used to assess the potency and mechanism of action of a novel ortho-phenoxylanilide ...[more]

PMID: 28608537

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Project description:Background and purposePre-synaptic neurotransmitter release is largely dependent on Ca(2+) entry through P/Q-type (Ca(V)2.1) voltage-gated Ca(2+) channels (PQCCs) at most mammalian, central, fast synapses. Barbiturates are clinical depressants and inhibit pre-synaptic Ca(2+) entry. PQCC barbiturate pharmacology is generally unclear, specifically in man. The pharmacology of the barbiturate pentobarbital (PB) in human recombinant alpha(1A) PQCCs has been characterized.Experimental approachPB effects on macroscopic Ca(2+)(I(Ca)) and Ba(2+)(I(Ba)) currents were studied using whole-cell patch clamp recording in HEK-293 cells heterologously expressing (alpha(1A))(human)(beta(2a)alpha(2)delta-1)(rabbit) PQCCs.Key resultsPB reversibly depressed peak current (I(peak)) and enhanced apparent inactivation (fractional current at 800 ms, r(800)) in a concentration-dependent fashion irrespective of charge carrier (50% inhibitory concentration: I(peak), 656 microM; r(800), 104 microM). Rate of mono-exponential I(Ba) decay was linearly dependent on PB concentration. PB reduced channel availability by deepening non-steady-state inactivation curves without altering voltage dependence, slowed recovery from activity-induced unavailable states and produced use-dependent block. PB (100 microM) induced use-dependent block during physiological, high frequency pulse trains and overall depressed PQCC activity by two-fold.Conclusion and implicationsThe results support a PB pharmacological mechanism involving a modulated receptor with preferential slow, bimolecular, open channel block (K(d)= 15 microM). Clinical PB concentrations (<200 microM) inhibit PQCC during high frequency activation that reduces computed neurotransmitter release by 16-fold and is comparable to the magnitude of Ca(2+)-dependent facilitation, G-protein modulation and intrinsic inactivation that play critical roles in PQCC modulation underlying synaptic plasticity. The results are consistent with the hypothesis that PB inhibition of PQCCs contributes to central nervous system depression underlying anticonvulsant therapy and general anaesthesia.

Project description:Arachidonic acid (AA) inhibits the activity of several different voltage-gated Ca(2+) channels by an unknown mechanism at an unknown site. The Ca(2+) channel pore-forming subunit (Ca(V)alpha(1)) is a candidate for the site of AA inhibition because T-type Ca(2+) channels, which do not require accessory subunits for expression, are inhibited by AA. Here, we report the unanticipated role of accessory Ca(V)beta subunits on the inhibition of Ca(V)1.3b L-type (L-) current by AA. Whole cell Ba(2+) currents were measured from recombinant channels expressed in human embryonic kidney 293 cells at a test potential of -10 mV from a holding potential of -90 mV. A one-minute exposure to 10 microM AA inhibited currents with beta(1b), beta(3), or beta(4) 58, 51, or 44%, respectively, but with beta(2a) only 31%. At a more depolarized holding potential of -60 mV, currents were inhibited to a lesser degree. These data are best explained by a simple model where AA stabilizes Ca(V)1.3b in a deep closed-channel conformation, resulting in current inhibition. Consistent with this hypothesis, inhibition by AA occurred in the absence of test pulses, indicating that channels do not need to open to become inhibited. AA had no effect on the voltage dependence of holding potential-dependent inactivation or on recovery from inactivation regardless of Ca(V)beta subunit. Unexpectedly, kinetic analysis revealed evidence for two populations of L-channels that exhibit willing and reluctant gating previously described for Ca(V)2 channels. AA preferentially inhibited reluctant gating channels, revealing the accelerated kinetics of willing channels. Additionally, we discovered that the palmitoyl groups of beta(2a) interfere with inhibition by AA. Our novel findings that the Ca(V)beta subunit alters kinetic changes and magnitude of inhibition by AA suggest that Ca(V)beta expression may regulate how AA modulates Ca(2+)-dependent processes that rely on L-channels, such as gene expression, enzyme activation, secretion, and membrane excitability.

Dataset Information

Inhibition of human N- and T-type calcium channels by an ortho-phenoxyanilide derivative, MONIRO-1.

Publications

Inhibition of human N- and T-type calcium channels by an ortho-phenoxyanilide derivative, MONIRO-1.

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