Oxygen (O(2)) is a prerequisite for cellular respiration in aerobic organisms but also elicits toxicity. To understand how animals cope with the ambivalent physiological nature of O(2), it is critical to elucidate the molecular mechanisms responsible for O(2) sensing. Here our systematic evaluation of transient receptor potential (TRP) cation channels using reactive disulfides with different redox potentials reveals the capability of TRPA1 to sense O(2). O(2) sensing is based upon disparate processes: whereas prolyl hydroxylases (PHDs) exert O(2)-dependent inhibition on TRPA1 activity in normoxia, direct O(2) action overrides the inhibition via the prominent sensitivity of TRPA1 to cysteine-mediated oxidation in hyperoxia. Unexpectedly, TRPA1 is activated through relief from the same PHD-mediated inhibition in hypoxia. In mice, disruption of the Trpa1 gene abolishes hyperoxia- and hypoxia-induced cationic currents in vagal and sensory neurons and thereby impedes enhancement of in vivo vagal discharges induced by hyperoxia and hypoxia. The results suggest a new O(2)-sensing mechanism mediated by TRPA1.

TRPA1 is a member of the transient receptor potential (TRP) cation channel family, and is predominantly expressed in nocicep-In TRPA1 responses to other cysteine-reactive inflammatory mediators, such as NO and H 2 O 2 , the extent of impairment by respective cysteine mutations differed from those in TRPA1 responses to 15d-PGJ 2 . Interestingly, the Cys421 mutation critically impaired the TRPA1 response to H + as well. Our findings suggest that TRPA1 channels are targeted by an array of inflammatory mediators to elicit inflammatory pain in the nervous system.

A primary mechanism for initiating smooth muscle contraction involves an increase in [Ca 2ϩ ] i , leading to myosin regulatory light chain (RLC) phosphorylation, crossbridge cycling, and force development (1, 2). Phosphorylation of Ser-19 on RLC of myosin II changes the orientation of myosin crossbridges, allowing actin activation of myosin ATPase activity. A similar mechanism occurs with nonmuscle myosin II with effects on many cellular actomyosin-dependent functions. The Ca 2ϩ -dependent phosphorylation of RLC is mediated by Ca 2ϩ ͞cal-modulin (CaM)-dependent myosin light chain kinase (MLCK), whereas myosin light chain phosphatase dephosphorylates RLC. In smooth muscles, agonists stimulate greater RLC phosphorylation and force than do depolarizing stimuli at comparable [Ca 2ϩ ] i because of Ca We developed a different CaM-sensor MLCK capable of monitoring MLCK activation to obtain temporal and quantitative information on Ca 2ϩ ͞CaM binding to MLCK where Ca 2ϩ -dependent CaM binding increased kinase activity coincident with a decrease in FRET (11). The CaM-sensor MLCK was expressed in smooth muscle tissues of transgenic mice to obtain quantitative insights on CaM activation of MLCK relative to [Ca 2ϩ ] i and RLC phosphorylation and force development. These results show that genetically encoded biosensors may be used to investigate physiological processes in tissues of transgenic mice. MethodsConstruction of SM8 35 KCS Plasmid. The pSM8 35 KCS construct was prepared by subcloning the 1.6-kb cDNA of Ca 2ϩ ͞CaM-sensor containing the MLCK CaM-binding sequence flanked by enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) (12) into the pSM8-CAT vector, which contains the smooth muscle ␣-actin promoter (13, 14). The 3.1-kb cDNA fragment of short rabbit smooth muscle MLCK was further subcloned into the site between the Ca 2ϩ ͞ CaM-sensor gene and the smooth muscle ␣-actin promoter in pSM8-CAT vector to produce pSM8 35 KCS construct. Correct This paper was submitted directly (Track II) to the PNAS office.

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca(2+) concentration ([Ca(2+)](i)). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca(2+)](i) with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.