State-Dependent Inhibition of Voltage-Gated Sodium Channels in Neuroblastoma Neuro-2A Cells by Arachidonic Acid from Halichondria okadai .

Voltage-gated sodium channels (Na v ) are closely associated with epilepsy, cardiac and skeletal muscle diseases, and neuropathic pain. Several toxic compounds have been isolated from the marine sponge Halichondria okadai ; however, toxic substances that modulate Na v are yet to be identified. This study aimed to identify Na v inhibitors from two snake venoms and H. okadai using mouse neuroblastoma Neuro-2A cells (N2A), which primarily express the specific Na v subtype Na v 1.7, using whole-cell patch-clamp recordings. We successfully isolated arachidonic acid (AA, 1 ) from the hexane extract of H. okadai , and then the fatty acid-mediated modulation of Na v in N2A was investigated in detail for the first time. Octanoic acid ( 2 ), palmitic acid ( 3 ), and oleic acid ( 4 ) showed no inhibitory activity at 100 μM, whereas AA ( 1 ), dihomo-γ-linolenic acid (DGLA, 5 ), and eicosapentaenoic acid (EPA, 6 ) showed IC 50 values of 6.1 ± 2.0, 58 ± 19, and 25 ± 4.0 μM, respectively ( N = 4, mean ± SEM). Structure and activity relationships were investigated for the first time using two ω-3 polyunsaturated fatty acids (PUFAs), EPA ( 6 ) and eicosatetraenoic acid (ETA, 7 ), and two ω-6 PUFAs, AA ( 1 ) and DGLA ( 5 ), to determine their effects on a resting state, activated state, and inactivated state. Steady-state analysis showed that the half inactivation potential was largely hyperpolarized by 10 μM AA ( 1 ), while 50 μM DGLA ( 5 ), 50 μM EPA ( 6 ), and 10 μM ETA ( 7 ) led to a slight change. The percentages of the resting state block were 24 ± 1, 22 ± 1, 34 ± 4, and 38 ± 9% in the presence of AA ( 1 ), DGLA ( 5 ), EPA ( 6 ), and ETA ( 7 ), respectively, with EPA ( 6 ) and ETA ( 7 ) exhibiting a greater inhibition than both AA ( 1 ) and DGLA ( 5 ), and their inhibitions did not increase in the following depolarization pulses. None of the compounds exhibited the use-dependent block. The half recovery times from the inactivated state for the control, AA ( 1 ), DGLA ( 5 ), EPA ( 6 ), and ETA ( 7 ) were 7.67 ± 0.33, 34.3 ± 1.10, 15.5 ± 1.10, 10.7 ± 0.31, and 3.59 ± 0.18 ms, respectively, with AA ( 1 ) exhibiting a distinctively large effect. Overall, distributed binding to the resting and the inactivated states of Na v would be significant for the inhibition of Na v , which presumably depends on the active structure of each PUFA.