Early detection of an O 2 deficit in the bloodstream is essential to initiate corrective changes in the breathing pattern of mammals. Carotid bodies serve an essential role in this respect; their type I cells depolarize when O 2 levels fall, causing voltage-gated Ca 2؉ entry. Subsequent neurosecretion elicits increased afferent chemosensory fiber discharge to induce appropriate changes in respiratory function (1). Although depolarization of type I cells by hypoxia is known to arise from K ؉ channel inhibition, the identity of the signaling pathway has been contested, and the coupling mechanism is unknown (2). We tested the hypothesis that AMP-activated protein kinase (AMPK) is the effector of hypoxic chemotransduction. AMPK is co-localized at the plasma membrane of type I cells with O 2 -sensitive K ؉ channels. In isolated type I cells, activation of AMPK using 5-aminoimidazole-4-carboxamide riboside (AICAR) inhibited O 2 -sensitive K ؉ currents (carried by large conductance Ca 2؉ -activated (BK Ca ) channels and TASK (tandem pore, acidsensing potassium channel)-like channels, leading to plasma membrane depolarization, Ca 2؉ influx, and increased chemosensory fiber discharge. Conversely, the AMPK antagonist compound C reversed the effects of hypoxia and AICAR on type I cell and carotid body activation. These results suggest that AMPK activation is both sufficient and necessary for the effects of hypoxia. Furthermore, AMPK activation inhibited currents carried by recombinant BK Ca channels, whereas purified AMPK phosphorylated the ␣ subunit of the channel in immunoprecipitates, an effect that was stimulated by AMP and inhibited by compound C. Our findings demonstrate a central role for AMPK in stimulus-response coupling by hypoxia and identify for the first time a link between metabolic stress and ion channel regulation in an O 2 -sensing system.Chronic and intermittent deficits in O 2 supply to the body precipitate a variety of pathologies including dementia (3) and pulmonary hypertension (4). To develop effective therapies, it is necessary to understand the homeostatic mechanisms that monitor O 2 supply to the body and elicit corrective changes in respiratory and circulatory function to maintain O 2 levels. O 2 -sensitive ion channels, which were first identified in the carotid body type I cell, play a pivotal role in this respect and have now been reported in a diverse range of highly specialized O 2 -sensing tissues (5). Within the carotid body, clusters of type I cells lie in presynaptic contact with afferent sensory fibers, whose discharge increases in proportion to the degree of systemic arterial O 2 deficit, providing information concerning blood O 2 levels to the central respiratory centers (1, 2). This occurs subsequent to hypoxic inhibition of type I cell K ϩ channels, membrane depolarization, voltage-gated Ca 2ϩ influx (6), and consequent neurotransmitter release. For many years, there has existed compelling evidence that mitochondria serve an important role in O 2 sensing by type I cells (2, 7). Inde...

a b s t r a c tTwo-pore channels (TPCs or TPCNs) are novel members of the large superfamily of voltage-gated cation channels with slightly higher sequence homology to the pore-forming subunits of voltagegated Ca 2+ and Na + channels than most other members. Recent studies demonstrate that TPCs locate to endosomes and lysosomes and form Ca 2+ release channels that respond to activation by the Ca 2+ mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). With multiple endolysosomal targeted NAADP receptors now identified, important new insights into the regulation of endolysosomal function in health and disease will therefore be unveiled. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.1. Two-pore channels represent an evolutionary link between the single and four pore-domain architectures of voltage-gated cation channelsIon channels play pivotal roles in signal transduction. Although many of the initial discoveries concerning ion channel functions were made in excitable cells, revealing the importance of these protein complexes in such essential activities as electric signal conduction and synaptic transmission in nerves, contraction of muscles, and hormone secretion of the endocrine system, it is important to know that ion channels are indispensable for all cells, including non-excitable cell types such as hepatocytes, adipocytes, keratinocytes, blood cells, endothelial and epithelial cells. Essentially, these channels are passages for ions to cross the lipid barriers of the plasma membrane and the membranes of intracellular organelles. These are tightly regulated processes and the direction of ionic movement is governed by the electrochemical gradients of the ion across a given membrane. For the majority of cells, the ionic movement only concerns Na + , K + , Ca 2+ , and Cl À because these are the major ions in the intracellular milieu and extracellular fluid. While Na + , K + and Cl À play critical roles in regulating membrane potential and, to some extent, substance transport, Ca 2+ is pivotal for cell signaling (see later). Depending on their ion selectivity, the ion channels have been designated as Na + , K + , Ca 2+ , Cl À channels or non-selective cation channels. Based on the mode of activation, they are also called voltage-gated, ligand-gated, Ca 2+ -activated, second messenger-operated, etc.Building on the early physiological and pharmacological studies, in the past three decades molecular cloning and sequence analyses have revealed a large number of ion channel proteins, which are not always evolutionarily related. Among them, the voltagegated K + , Ca 2+ and Na + channels are evolutionarily linked [1]. The ion-conducting pore of the K + channels is formed by four poreforming subunits, which are either identical or distinct but share sequence homology. Each subunit contains six transmembrane (TM) a helical segments that can be divided into two parts. The first four (S1-S4) are involved in channel regulation including voltage-sen...

Two-pore segment channel 2 (TPC2) is a ubiquitously expressed, lysosomally targeted ion channel that aids in terminating autophagy and is inhibited upon its association with mechanistic target of rapamycin (mTOR). It is controversial whether TPC2 mediates lysosomal Ca2+ release or selectively conducts Na+ and whether the binding of nicotinic acid adenine dinucleotide phosphate (NAADP) or phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is required for the activity of this ion channel. We show that TPC2 is required for intracellular Ca2+ signaling in response to NAADP or to mTOR inhibition by rapamycin. In pulmonary arterial myocytes, rapamycin and NAADP evoked global Ca2+ transients that were blocked by depletion of lysosomal Ca2+ stores. Preincubation of cells with high concentrations of rapamycin resulted in desensitization and blocked NAADP-evoked Ca2+ signals. Moreover, rapamycin and NAADP did not evoke discernable Ca2+ transients in myocytes derived from Tpcn2 knockout mice, which showed normal responses to other Ca2+-mobilizing signals. In HEK293 cells stably overexpressing human TPC2, shRNA-mediated knockdown of mTOR blocked rapamycin- and NAADP-evoked Ca2+ signals. Confocal imaging of a genetically encoded Ca2+ indicator fused to TPC2 demonstrated that rapamycin-evoked Ca2+ signals localized to lysosomes and were in close proximity to TPC2. Therefore, inactivation of mTOR may activate TPC2 and consequently lysosomal Ca2+ release.

HPV supports ventilation-perfusion matching in the lung by diverting blood flow away from oxygen-deprived areas towards regions rich in O2. However, in diseases such as emphysema and cystic fibrosis, widespread HPV leads to hypoxic pulmonary hypertension and ultimately right heart failure. Determining the precise mechanism(s) that underpins hypoxia-response coupling will therefore advance understanding of the fundamental processes contributing to related pathophysiology and provide for improved therapeutics.

Vital homeostatic mechanisms monitor O2 supply and adjust respiratory and circulatory function to meet demand. The pulmonary arteries and carotid bodies are key systems in this respect. Hypoxic pulmonary vasoconstriction (HPV) aids ventilation-perfusion matching in the lung by diverting blood flow from areas with an O2 deficit to those rich in O2, while a fall in arterial pO2 increases sensory afferent discharge from the carotid body to elicit corrective changes in breathing patterns. We discuss here the new concept that hypoxia, by inhibiting oxidative phosphorylation, activates AMP-activated protein kinase (AMPK) leading to consequent phosphorylation of target proteins, such as ion channels, which initiate pulmonary artery constriction and carotid body activation. Consistent with this view, AMPK knockout mice exhibit an impaired ventilatory response to hypoxia. Thus, AMPK may be sufficient and necessary for hypoxia-response coupling and may regulate O2 and thereby energy (ATP) supply at the whole body as well as the cellular level.