Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca2+]i) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca2+]i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (KCa2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of KCa2.2 channels within 3 h after the onset of glutamate exposure. Activation of KCa2 channels preserved KCa2 expression and significantly reduced pathological increases in [Ca2+]i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for KCa2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders.

Small-conductance Ca2+-activated K+ channel activation is an emerging therapeutic approach for treatment of neurological diseases, including stroke, amyotrophic lateral sclerosis and schizophrenia. Our previous studies showed that activation of SK channels exerted neuroprotective effects through inhibition of NMDAR-mediated excitotoxicity. In this study, we tested the therapeutic potential of SK channel activation of NS309 (25 μM) in cultured human postmitotic dopaminergic neurons in vitro conditionally immortalized and differentiated from human fetal mesencephalic cells. Quantitative RT-PCR and western blotting analysis showed that differentiated dopaminergic neurons expressed low levels of SK2 channels and high levels of SK1 and SK3 channels. Further, protein analysis of subcellular fractions revealed expression of SK2 channel subtype in mitochondrial-enriched fraction. Mitochondrial complex I inhibitor rotenone (0.5 μM) disrupted the dendritic network of human dopaminergic neurons and induced neuronal death. SK channel activation reduced mitochondrial membrane potential, while it preserved the dendritic network, cell viability and ATP levels after rotenone challenge. Mitochondrial dysfunction and delayed dopaminergic cell death were prevented by increasing and/or stabilizing SK channel activity. Overall, our findings show that activation of SK channels provides protective effects in human dopaminergic neurons, likely via activation of both membrane and mitochondrial SK channels. Thus, SK channels are promising therapeutic targets for neurodegenerative disorders such as Parkinson's disease, where dopaminergic cell loss is associated with progression of the disease.

The iminodihydroquinoline WIN 17317-3 was previously shown to inhibit selectively the voltage-gated potassium channels, K(v)1.3 and K(v)1.4 [Hill, R. J., et al. (1995) Mol. Pharmacol. 48, 98-104; Nguyen, A., et al. (1996) Mol. Pharmacol. 50, 1672-1679]. Since these channels are found in brain, radiolabeled WIN 17317-3 was synthesized to probe neuronal K(v)1 channels. In rat brain synaptic membranes, [(3)H]WIN 17317-3 binds reversibly and saturably to a single class of high-affinity sites (K(d) 2.2 +/- 0.3 nM; B(max) 5.4 +/- 0.2 pmol/mg of protein). However, the interaction of [(3)H]WIN 17317-3 with brain membranes is not sensitive to any of several well-characterized potassium channel ligands. Rather, binding is modulated by numerous structurally unrelated sodium channel effectors (e.g., channel toxins, local anesthetics, antiarrhythmics, and cardiotonics). The potency and rank order of effectiveness of these agents in affecting [(3)H]WIN 17317-3 binding is consistent with their known abilities to modify sodium channel activity. Autoradiograms of rat brain sections indicate that the distribution of [(3)H]WIN 17317-3 binding sites is in excellent agreement with that of sodium channels. Furthermore, WIN 17317-3 inhibits sodium currents in CHO cells stably transfected with the rat brain IIA sodium channel with high affinity (K(i) 9 nM), as well as agonist-stimulated (22)Na uptake in this cell line. WIN 17317-3 interacts similarly with skeletal muscle sodium channels but is a weaker inhibitor of the cardiac sodium channel. Together, these results demonstrate that WIN 17317-3 is a new, high-affinity, subtype-selective ligand for sodium channels and is a potent blocker of brain IIA sodium channels.

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