Testosterone modulates seizure susceptibility, but the underlying mechanisms are obscure. Recently, we demonstrated that testosterone affects seizure activity via its conversion to neurosteroids in the brain. Androstanediol (5␣-androstan-3␣,17␤-diol) is an endogenous neurosteroid synthesized from testosterone. However, the molecular mechanism underlying the seizure protection activity of androstanediol remains unclear. Here, we show that androstanediol has positive allosteric activity as a GABA A receptor modulator. In whole-cell recordings from acutely dissociated hippocampus CA1 pyramidal cells, androstanediol (but not its 3␤-epimer) produced a concentration-dependent enhancement of GABA-activated currents (EC 50 of 5 M). At 1 M, androstanediol produced a 50% potentiation of GABA responses. In the absence of GABA, androstanediol has moderate direct effects on GABA A receptor-mediated currents at high concentrations. Systemic doses of androstanediol (5-100 mg/kg), but not its 3␤-epimer, caused dose-dependent suppression of behavioral and electrographic seizures in mouse hippocampus kindling, which is a model of temporal lobe epilepsy. The ED 50 value for antiseizure effects of androstanediol was 50 mg/kg, which did not produce sedation/motor toxicity. At high (2ϫ ED 50 ) doses, androstanediol produced complete seizure protection that lasted for up to 3 h after injection. The estimated plasma concentrations of androstanediol producing 50% seizure protection in the kindling model (10.6 M) are within the range of concentrations that modulate GABA A receptors. These studies suggest that androstanediol could be a neurosteroid mediator of testosterone actions on neuronal excitability and seizure susceptibility via its activity as a GABA A receptor modulator and that androstanediol may play a key role in men with epilepsy, especially during the age-related decline in androgen levels.

The daily rhythm of L‐type voltage‐gated calcium channels (L‐VGCCs) is part of the cellular mechanism underlying the circadian regulation of retina physiology and function. However, it is not completely understood how the circadian clock regulates L‐VGCC current amplitudes without affecting channel gating properties. The phosphatidylinositol 3 kinase–protein kinase B (PI3K–Akt) signaling pathway has been implicated in many vital cellular functions especially in trophic factor‐induced ion channel trafficking and membrane insertion. Here, we report that PI3K–Akt signaling participates in the circadian phase‐dependent modulation of L‐VGCCs. We found that there was a circadian regulation of Akt phosphorylation on Thr308 that peaked at night. Inhibition of PI3K or Akt significantly decreased L‐VGCC current amplitudes and the expression of membrane‐bound L‐VGCCα1D subunit only at night but not during the subjective day. Photoreceptors transfected with a dominant negative Ras had significantly less expression of phosphorylated Akt and L‐VGCCα1D subunit compared with non‐transfected photoreceptors. Interestingly, both PI3K–Akt and extracellular signal‐related kinase were downstream of Ras, and they appeared to be parallel and equally important pathways to regulate L‐VGCC rhythms. Inhibition of either pathway abolished the L‐VGCC rhythm indicating that there were multiple mechanisms involved in the circadian regulation of L‐VGCC rhythms in retina photoreceptors.

The L-type voltage-gated calcium channels (L-VGCCs) are activated under high depolarization voltages. They are vital for diverse biological events, including cell excitability, differentiation, and synaptic transmission. In retinal photoreceptors, L-VGCCs are responsible for neurotransmitter release and are under circadian influences. However, the mechanism of L-VGCC regulation in photoreceptors is not fully understood. Here, we show that retinoschisin, a highly conserved extracellular protein, interacts with the L-VGCC␣1D subunit and regulates its activities in a circadian manner. Mutations in the gene encoding retinoschisin (RS1) cause retinal disorganization that leads to early onset of macular degeneration. Since ion channel activities can be modulated through interactions with extracellular proteins, disruption of these interactions can alter physiology and be the root cause of disease states. Co-immunoprecipitation and mammalian two-hybrid assays showed that retinoschisin and the N-terminal fragment of the L-VGCC␣1 subunit physically interacted with one another. The expression and secretion of retinoschisin are under circadian regulation with a peak at night and nadir during the day. Inhibition of L-type VGCCs decreased membrane-bound retinoschisin at night. Overexpression of a missense RS1 mutant gene, R141G, into chicken cone photoreceptors caused a decrease of L-type VGCC currents at night. Our findings demonstrate a novel bidirectional relationship between an ion channel and an extracellular protein; L-type VGCCs regulate the circadian rhythm of retinoschisin secretion, whereas secreted retinoschisin feeds back to regulate L-type VGCCs. Therefore, physical interactions between L-VGCC␣1 subunits and retinoschisin play an important role in the membrane retention of L-VGCC␣1 subunits and photoreceptor-bipolar synaptic transmission.

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Exposure to ethanol levels reached in circulation during alcohol intoxication (≥10 mM) constricts cerebral arteries in rats and humans. Remarkably, targets and mechanisms underlying this action remain largely unidentified. Artery diameter is regulated by myocyte Ca2+ sparks, a vasodilatory signal contributed to by type 2 ryanodine receptors (RyR2). Using laser confocal microscopy in rat cerebral arteries and bilayer electrophysiology we unveil that ethanol inhibits both Ca2+ spark and RyR2 activity with IC50<20 mM, placing RyR2 among the ion channels that are most sensitive to ethanol. Alcohol directly targets RyR2 and its lipid microenvironment, leading to stabilization of RyR2 closed states.

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Jian K, Barhoumi R, Ko ML, Ko GY. Inhibitory effect of somatostatin-14 on L-type voltage-gated calcium channels in cultured cone photoreceptors requires intracellular calcium. J Neurophysiol 102: 1801-1810, 2009. First published July 15, 2009 doi:10.1152/jn.00354.2009. The inhibitory effects of somatostatin have been well documented for many physiological processes. The action of somatostatin is through G-protein-coupled receptor-mediated second-messenger signaling, which in turn affects other downstream targets including ion channels. In the retina, somatostatin is released from a specific class of amacrine cells. Here we report that there was a circadian phase-dependent effect of somatostatin-14 (SS14) on the L-type voltage-gated calcium channels (LVGCCs) in cultured chicken cone photoreceptors, and our study reveals that this process is dependent on intracellular calcium stores. Application of 500 nM SS14 for 2 h caused a decrease in L-VGCC currents only during the subjective night but not the subjective day. We then explored the cellular mechanisms underlying the circadian phase-dependent effect of SS14. The inhibitory effect of SS14 on L-VGCCs was mediated through the pertussis-toxin-sensitive Gprotein-dependent somatostatin receptor 2 (sst2). Activation of sst2 by SS14 further activated downstream signaling involving phospholipase C and intracellular calcium stores. Mobilization of intracellular Ca 2ϩ was required for somatostatin induced inhibition of photoreceptor L-VGCCs, suggesting that somatostatin plays an important role in the modulation of photoreceptor physiology.