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

초록

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 approximate to 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(Delta C36-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* ( R50G(NL)(& lowast; )) activity showed similar to 100-fold lower ATP sensitivity (K-1/2 approximate to 2.0 mM) than that of the wild-type K-ATP (K-1/2 approximate to 20 mu M). 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 R50G(NL)(& lowast;) activity in crammed CB cells, [ATP](cyt) was estimated to be similar to 4.9 mM in normoxia. In CB cells, hypoxia (1% O-2, 15 s) did not affect R50G(NL)(& lowast;) activity, indicating that [ATP](cyt) remained stable during hypoxia. FCCP (1 mu M), a mitochondrial uncoupler, increased R50G(NL)(& lowast;) activity, which corresponded to [ATP](cyt) of similar to 3.7 mM. As hypoxia (similar to 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.

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