Whole cell patch-clamp recordings of rat midbrain dopaminergic neurons isolate a sulphonylurea- and ATP-sensitive component of potassium currents activated by hypoxia

The effects of brief (2-4 min) hypoxia on presumed dopaminergic "principal" neurons of the rat ventral mesencephalon were investigated by using either intracellular or whole cell patch-clamp recordings in in vitro conditions. Under single-electrode voltage clamp, with sharp microelectrode (V-h -60 mV), a brief hypoxia caused an outward current (hypo(OUT)) of 110.2 +/- 15.2 (SE) pA (n = 18),which was followed by a posthypoxic outward current (posthypo(OUT)) of 149.6 +/- 10.6 pA (n = 18). Although the hypo(OUT) reversed at -83.7 +/- 3.8 mV (n = 18), the posthypo(OUT) did not reverse. The K-ATP(+)-blocking sulphonylureas tolbutamide (100 mu M) and glibenclamide (30 mu M), significantly reduced the peak of the hypo(OUT) by 47.6 +/- 7.7% (n = 16) and 54.18 +/- 7.5% (n = 3), respectively. In contrast, they did not affect the posthypo(OUT). Extracellular barium (300 mu M to 1 mM) almost abolished the hypo(OUT), leaving the posthypo(OUT) unchanged. The large K+ channel blocker charybdotoxin (10-50 nM), depressed the hypo(OUT) after tolbutamide treatment. To investigate whether or not cytosolic factors might control the development of the hypo(OUT), we dialyzed the principal neurons by patch-clamp recordings (V-h -60 mV). Under whole cell recordings hypoxia evoked an hypo(OUT) of 70.2 +/- 14.5 pA that reversed polarity at -87.9 +/- 5.1 mV (n = 8). A small posthypoxic response was detected upon reoxygenation in a few neurons (4 out of 14). Three different sulphonylureas, tolbutamide (100 mu M), glibenclamide (10-30 mu M), and glipizide (100 nM) completely blocked the hypo(OUT) in patch-clamped neurons. The hypo(OUT) was also abolished by extracellular BaCl2 (300 mu M). When the content of ATP in the dialyzate was raised from 2 to 10 mM no outward current/hyperpolarization was evoked by hypoxia. These data suggest that the hypo(OUT), in principal neurons, is a complex response sustained by at least two barium-sensitive components: 1) an ATP-dependent, sulphonylurea-sensitive K+ conductance which could be isolated by the patch-clamp techniques and 2) a K+ conductance remaining after tolbutamide in intracellularly recorded neurons, which is sensitive to charybdotoxin and dependent on dialyzable cytosolic factors.