BPS2026 - Acute hypoxia inhibits K2p3/9 without reducing cytosolic [ATP] in carotid body glomus cells

Hypoxia stimulates ventilation through a chemoreflex mechanism that begins within the carotid body glomus (CB) cells. In CB cells, hypoxia inhibits O 2 -sensitive background K 2P 3/9 channels (TASK-1/3) and increases Ca 2+ oscillations, which initiate the chemosensory response. The mechanism of K 2P 3/9 inhibition remains unclear. Because K 2P 3/9 is also modulated by cytosolic ATP (K 1/2 , ∼2.5 mM), it has been hypothesized that hypoxia inhibits K 2P 3/9 by reducing mitochondrial ATP production and cytosolic ATP concentration ([ATP] cyt ). To test this hypothesis, we developed a K ATP channel (Kir6/SUR)-based bioassay to measure [ATP] cyt in CB cells. Co-expression of Kir6.2(ΔC36-R50G) and SUR1(G1485D) mutant subunits in HEK293 cells formed a functional K ATP channel (referred to as R50G). Normalized R50G (R50G NL )-[ATP] cyt relationship obtained in inside-out patches showed ∼100-fold lower ATP sensitivity (K 1/2 , ∼2.0 mM) than the wildtype K ATP (K 1/2 , ∼20 μM). To determine [ATP] cyt in CB cells, an inside-out patch from a HEK293 cell expressing R50G was crammed into a CB cell, exposing the patch to the CB cell cytoplasm. In normoxia (21% O 2 , 37°C), R50G NL activity in crammed patches was 0.12 ± 0.3, which corresponded to [ATP] cyt of ∼5.0 mM. When the same cell with the crammed patch was exposed to hypoxia (∼1% O 2 , ∼15 s), R50G NL activity in the patch was 0.13 ± 0.3, indicating that [ATP] cyt in CB cells remained stable at ∼5.0 mM during acute hypoxia. The same level of hypoxia inhibited K 2P 3/9 by 65% within 5 s. Based on the [ATP]-K 2P 3/9 relationship, a 65% inhibition of K 2P 3/9 is achieved by a ∼60% decrease in [ATP] cyt (from 5 mM to 2 mM), which is expected to cause a ∼360% increase in R50G NL activity. Our study suggests that inhibition of K 2P 3/9 by acute hypoxia is unlikely to be mediated by a reduction of [ATP] cyt in CB cells.