Acute Hypoxia Inhibits <scp> K _2P 3 </scp> /9 ( <scp>TASK1</scp> /3) Independently of Cytosolic <scp>ATP</scp> in Carotid Body Type 1 Cells

ABSTRACT Hypoxia stimulates ventilation through a chemosensory reflex that is initiated by inhibition of the O 2 ‐sensitive K + channels such as K 2P 3/9 in carotid body Type 1 glomus (CB) cells. Because K 2P 3/9 activity is sensitive to cytosolic ATP concentration ([ATP] cyt , K 1/2 ≈ 2.5 mM), it has been proposed that hypoxia suppresses mitochondrial ATP production and thereby reduces [ATP] cyt to inhibit K 2P 3/9 activity. To test this hypothesis, we developed a K ATP channel‐based bioassay using a patch‐cramming approach to measure [ATP] cyt in living cells. Co‐expression of Kir6.2(ΔC36‐R50G) and SUR1(G1485D) in HEK293 cells formed a functional K ATP channel (designated R50G*) with markedly reduced ATP sensitivity. In inside‐out patches, normalized R50G* () activity showed ~100‐fold lower ATP sensitivity (K 1/2 ≈ 2.0 mM) than that of the wild‐type K ATP (K 1/2 ≈ 20 μM). Inside‐out patches from HEK293 cells containing R50G* were crammed into CB cells under normoxia (21% O 2 ), exposing the patch to the CB cell cytoplasm. Based on activity in crammed CB cells, [ATP] cyt was estimated to be ~4.9 mM in normoxia. In CB cells, hypoxia (1% O 2 , 15 s) did not affect activity, indicating that [ATP] cyt remained stable during hypoxia. FCCP (1 μM), a mitochondrial uncoupler, increased activity, which corresponded to [ATP] cyt of ~3.7 mM. As hypoxia (~1% O 2 ) inhibited K 2P 3/9 activity by 65% within 5 s, our result demonstrates that inhibition of K 2P 3/9 by hypoxia is independent of [ATP] cyt and that ATP depletion is unlikely to be the initiating signal for hypoxia‐induced excitation of CB cells.