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

Voltage-gated sodium channels (Nav) 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 Nav are yet to be identified. This study aimed to identify Nav inhibitors from two snake venoms and H. okadai using mouse neuroblastoma Neuro-2A cells (N2A), which primarily express the specific Nav subtype Nav1.7, 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 Nav 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 mu M, whereas AA (1), dihomo-y-linolenic acid (DGLA, 5), and eicosapentaenoic acid (EPA, 6) showed IC50 values of 6.1 +/- 2.0, 58 +/- 19, and 25 +/- 4.0 mu M, respectively (N = 4, mean +/- SEM). Structure and activity relationships were investigated for the first time using two co-3 polyunsaturated fatty acids (PUFAs), EPA (6) and eicosatetraenoic acid (ETA, 7), and two co-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 mu M AA (1), while 50 mu M DGLA (5), 50 mu M EPA (6), and 10 mu M 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 Nav would be significant for the inhibition of Nav, which presumably depends on the active structure of each PUFA.