Acute Hypoxia Inhibits K2P3/9 (TASK1/3) Independently of Cytosolic ATP in Carotid Body Type 1 Cells.

Hypoxia stimulates ventilation through a chemosensory reflex that is initiated by inhibition of the O2-sensitive K+ channels such as K2P3/9 in carotid body Type 1 glomus (CB) cells. Because K2P3/9 activity is sensitive to cytosolic ATP concentration ([ATP]cyt, K1/2 ≈ 2.5 mM), it has been proposed that hypoxia suppresses mitochondrial ATP production and thereby reduces [ATP]cyt to inhibit K2P3/9 activity. To test this hypothesis, we developed a KATP 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 KATP channel (designated R50G*) with markedly reduced ATP sensitivity. In inside-out patches, normalized R50G* ( R 50 G NL * ) activity showed ~100-fold lower ATP sensitivity (K1/2 ≈ 2.0 mM) than that of the wild-type KATP (K1/2 ≈ 20 μM). Inside-out patches from HEK293 cells containing R50G* were crammed into CB cells under normoxia (21% O2), exposing the patch to the CB cell cytoplasm. Based on R 50 G NL * activity in crammed CB cells, [ATP]cyt was estimated to be ~4.9 mM in normoxia. In CB cells, hypoxia (1% O2, 15 s) did not affect R 50 G NL * activity, indicating that [ATP]cyt remained stable during hypoxia. FCCP (1 μM), a mitochondrial uncoupler, increased R 50 G NL * activity, which corresponded to [ATP]cyt of ~3.7 mM. As hypoxia (~1% O2) inhibited K2P3/9 activity by 65% within 5 s, our result demonstrates that inhibition of K2P3/9 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.