Mitochondrial KATP channels stabilize intracellular Ca2+ during hypoxia in retinal horizontal cells of goldfish (Carassius auratus).

Abstract

Neurons of the retina require oxygen to survive. In hypoxia, neuronal ATP production is impaired, ATP-dependent ion pumping is reduced, transmembrane ion gradients are dysregulated, and [Ca2+]i increases enough to trigger excitotoxic cell death. Central neurons of the common goldfish (Carassius auratus) are hypoxia-tolerant, but little is known about how goldfish retinas withstand hypoxia. To study the cellular mechanisms of hypoxia tolerance, we isolated retinal interneurons (horizontal cells; HCs), and measured intracellular Ca2+ concentration ([Ca2+]i) with Fura-2. Goldfish HCs maintained [Ca2+]i throughout 1 h of hypoxia, whereas [Ca2+]i increased irreversibly in HCs of the hypoxia-sensitive rainbow trout (Oncorhynchus mykiss) with just 20 min of hypoxia. Our results suggest mitochondrial ATP-dependent K+ channels (mKATP) are necessary to stabilize [Ca2+]i throughout hypoxia. In goldfish HCs, [Ca2+]i increased when mKATP was blocked with glibenclamide or 5-HD, whereas an mKATP agonist (diazoxide) prevented [Ca2+]i from increasing in hypoxia in trout HCs. We showed that hypoxia protects goldfish HCs via mKATP channels. Glycolytic inhibition with 2-deoxyglucose increased [Ca2+]i, which was rescued by hypoxia in an mKATP-dependent manner. We found no evidence of plasmalemmal KATP channels in patch-clamp experiments. Instead, we confirmed the involvement of KATP in mitochondria with TMRE imaging, as hypoxia rapidly (<5 min) depolarized mitochondria in an mKATP-sensitive manner. We conclude that mKATP channels initiate a neuroprotective pathway in goldfish HCs to maintain [Ca2+]i and avoid excitotoxicity in hypoxia. This model provides novel insight into the cellular mechanisms of hypoxia tolerance in the retina.