O2−sensitive K+ channels in immortalised rat chromaffin‐cell‐derived MAH cells

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Abstract
The regulation of K+ channels by O2 levels is a key link between hypoxia and neurotransmitter release in neuroendocrine cells. Here, we examined the effects of hypoxia on K+ channels in the immortalised v‐myc, adrenal‐derived HNK1+ (MAH) cell line. MAH cells possess a K+ conductance that is sensitive to Cd2+, iberiotoxin and apamin, and which is inhibited by ≈24 % when exposed to a hypoxic perfusate (O2 tension 20 mmHg). This conductance was attributed to high‐conductance Ca2+‐activated K+ (BK) and small‐conductance Ca2+‐activated K+ (SK) channels, which are major contributors to the O2‐sensitive K+ conductance in adrenomedullary chromaffin cells. Under low [Ca2+]i conditions that prevented activation of Ca2+‐dependent K+ conductances, a rapidly activating and slowly inactivating K+ conductance, sensitive to both TEA and 4‐aminopyridine (4‐AP), but insensitive to 100 nm charybdotoxin (CTX), was identified. This current was also reduced (by ≈25 %) when exposed to hypoxia. The hypoxia‐sensitive component of this current was greatly attenuated by 10 mm 4‐AP, but was only slightly reduced by 10 mm TEA. This suggests the presence of delayed‐rectifier O2‐sensitive channels comprising homomultimeric Kv1.5 or heteromultimeric Kv1.5/Kv1.2 channel subunits. The presence of both Kv1.5 and Kv1.2 α‐subunits was confirmed using immunocytochemical techniques. We also demonstrated that these K+ channel subunits are present in neonatal rat adrenomedullary chromaffin cells in situ. These data indicate that MAH cells possess O2‐sensitive K+ channels with characteristics similar to those observed previously in isolated chromaffin cells, and therefore provide an excellent model for examining the cellular mechanisms of O2 sensing in adrenomedullary chromaffin cells.