Neuroepithelial bodies act as airway oxygen sensors. The lung carcinoma line H146 is an established model for neuroepithelial body cells. Although O 2 sensing in both cells is via NADPH oxidase H 2 O 2 /free radical production and acute hypoxia promotes K ؉ channel closure and cell depolarization, the identity of the K ؉ channel is still controversial. However, recent data point toward the involvement of a member of the tandem P domain family of K ؉ channels. Reverse transcription-polymerase chain reaction screening indicates that all known channels other than hTWIK1 and hTRAAK are expressed in H146 cells. Our detailed pharmacological characterization of the O 2 -sensitive K ؉ current described herein is compatible with the involvement of hTASK1 or hTASK3 (pH dependence, tetraethylammonium and dithiothreitol insensitivity, blockade by arachidonic acid, and halothane activation). Furthermore, we have used antisense oligodeoxynucleotides directed against hTASK1 and hTASK3 to suppress almost completely the hTASK1 protein and show that these cells no longer respond to acute hypoxia; this behavior was not mirrored in liposome-only or missense-treated cells. Finally, we have used Zn 2؉ treatment as a maneuver able to discriminate between these two homologues of hTASK and show that the most likely candidate channel for O 2 sensing in these cells is hTASK3.

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The combination of studies in native tissues and immortalised model systems during the last decade has made possible a deeper understanding of the physiology and functional morphology of arterial and airway oxygen sensors. Complementary and overlapping information from these earlier studies has allowed a detailed description of the cellular events that link decreased environmental oxygen to the release of physiologically important vasoactive transmitters. Since these basic pathways have now been defined functionally, what remains to be determined is the molecular identity of the specific proteins involved in the signal transduction pathways, and how these proteins interact to produce a full physiological response. With these goals clearly in sight, we have embarked upon a strategy that is a novel combination of proteomics and functional genomics. It is hoped this strategy will enable us to develop and refine the initial models in order to understand more completely the process of oxygen sensing in health and disease. Anat Rec Part A 270A: 41-50, 2003.

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Various cardiorespiratory diseases (e.g. congestive heart failure, emphysema) result in systemic hypoxia and patients consequently demonstrate adaptive cellular responses which predispose them to conditions such as pulmonary hypertension and stroke. Central to many affected excitable tissues is activity of large conductance, Ca 2؉ -activated K ؉ (maxiK) channels. We have studied maxiK channel activity in HEK293 cells stably co-expressing the most widely distributed of the human ␣-and ␤-subunits that constitute these channel following maneuvers which mimic severe hypoxia. ] i could sustain an acute hypoxic inhibitory response. Chronic hypoxia caused no change in ␣-subunit immunoreactivity with Western blotting but evoked a 3-fold increase in ␤-subunit expression. These observations were fully supported by immunocytochemistry, which also suggested that chronic hypoxia augmented ␣/␤-subunit colocalization at the plasma membrane. Using a novel nuclear run-on assay and RNase protection we found that chronic hypoxia did not alter mRNA production rates or steady-state levels, which suggests that this important environmental cue modulates maxiK channel function via post-transcriptional mechanisms.Crucial to the cellular and physiological response to acute perturbation of systemic and/or pulmonary O 2 levels is the rapid inhibition of K ϩ channels by hypoxia (see Ref. 1 for recent review). Thus, acute modulation of ion channel activity is central to the homeostatic mechanisms that underlie chemosensing in carotid body (2-4), neuroepithelial body (5, 6) (and its immortalized cellular counterpart, H146 cells, Ref. 7) and (8 -11) systemic vascular smooth muscle (12). Although somewhat controversial, ion channel inhibition has also been implicated in hypoxic vasoconstriction in the pulmonary circulation (13).In addition, such O 2 sensitivity is believed to play a significant role in modulation of excitability in several cellular components of the mammalian nervous system (14 -17).Although O 2 -sensitive tissues express a wide variety of channel types, central to the cellular mechanism of acute O 2 sensing in several is hypoxic suppression of large conductance Ca 2ϩ -activated K ϩ (maxiK) channels. Indeed, hypoxic inhibition of native maxiK channel activity has been demonstrated in carotid body (4, 18, 19), pulmonary arteriolar smooth muscle (20), chromaffin cells (21), and central neurons (20,22). Although their contribution to carotid body, chromaffin cell, and central neuronal function is well supported, some controversy still surrounds their involvement in pulmonary vasoconstriction (15) and there is good evidence for both delayed rectifier (23) and tandem P domain K ϩ channels in the response (24); the latter observation is fully supported by our recent reports of O 2 sensitivity of the recombinant human tandem P domain channels, hTASK1 (25), and hTASK3 (26).Tissue specificity notwithstanding, we have recently demonstrated at the single channel level that a recombinant human maxiK channel can be rapidly and reversibly in...