MdHCLB channels expressed in Xenopus oocytes produced curren
MdHCLB channels expressed in Xenopus oocytes produced currents in response to HA and GABA (Fig. 1, Fig. 3). This is in agreement with the findings with Drosophila HCLB channels (Gisselmann et al., 2004). In the HA concentration-response curves, the effects of GABA were additive to those of HA only in the low HA concentration range (Fig. 4B), thereby indicating that GABA may act at the orthosteric site of MdHCLB channels. When considered with the findings that rat or human type-A GABARs responded to HA (Bianchi et al., 2011, Saras et al., 2008), our finding suggests that there is a similarity in agonist recognition at the orthosteric sites between HACls and GABACls. Recently, two residues, Asp163 in loop B and Arg120 in loop E, were predicted to be involved in the direct modulation of α1β3 GABARs by HA (Thiel et al., 2015). More studies are needed to identify amino Cinchonidine sale residues that are responsible for GABA and HA binding in MdHCLB channels. Drosophila HCLB channels, which are expressed in large ventral lateral neurons (1-LNvs) (Hong et al., 2006), were recently reported to be involved in wake-promoting signaling (Oh et al., 2013). GABACls composed of RDL subunits also have a critical role in the control of sleep or rest/arousal patterns by regulating the excitability of l-LNvs in the Drosophila brain (Chung et al., 2009, Liu et al., 2014, McCarthy et al., 2011, Parisky et al., 2008). It is interesting to note that wakefulness in mice is also controlled by HA and GABA cotransmission from the tuberomammillary nucleus of the hypothalamus (Yu et al., 2015), although the mechanism differs from that in insects. In the antennal lobe of M. sexta, the mesothoracic to deutocerebral histaminergic neurons (MDHns) arborize in ventral glomeruli. As the GABAergic local interneurons (LNs), which also ramify in the glomeruli, were labeled with an anti-HCLB antibody, it is speculated that the LNs are postsynaptic to MDHns (Bradley et al., 2016). Given the colocalization of HCLB channels and GABACls and the ability of GABA to cross-react with HCLB channels, it is possible that GABA spillover may affect inhibitory neurotransmission mediated by HCLB channels. We examined the effects of H1R, H2R and H3R agonists on MdHCLs. Although THA, an H1R agonist, was the most potent partial agonist of MdHCLB receptors among the three tested agonists, structural modifications resulted in drastic decreases in potency (Fig. 2). Biogenic amines, such as 5-HT, DA, OA and NA, showed no effects on native receptors in Musca LMCs (Hardie, 1988), but these amines evoked small currents in MdHCLB channels (Fig. 3A). 5-HT and DA also exhibited the competitive antagonism of MdHCLB channels (Fig. 5E). The responses of Musca LMCs to light were inhibited by nicotinic and muscarinic AChR antagonists (Hardie, 1988). Benzoquinonium, gallamine and atropine were among the most potent antagonists. In our experiments, the nicotinic AChR antagonist d-tubocurarine inhibited HA-induced currents more potently than GABA-induced currents in MdHCLB channels (Fig. 6A and B). The potent HA receptor antagonists in Musca LMCs were all H2R antagonists (Hardie, 1988). Consistent with this finding, cimetidine, an H2R antagonist, equipotently inhibited both HA- and GABA-induced currents in MdHCLB channels (Fig. 6A). Four LGCC antagonists (picrotoxinin, TBPS, fipronil and fluralaner), an LGCC activator (ivermectin B1a) and two insecticides did not show marked antagonism of MdHCLB channels. The 2′ Ala in TM2 and 36′ Gly in TM3 (highlighted in red in Fig. 7) is critical for the antagonism and activation of LGCCs (Nakao et al., 2013, Ozoe, 2013, Ozoe et al., 2015). In MdHCL channels, the amino acids at these positions are substituted with Thr and Ser, respectively. These substitutions are most likely responsible for the reduced effects of these LGCC antagonists and activators. In conclusion, we cloned cDNAs encoding two HACls, MdHCLA and MdHCLB channels, from the housefly M. domestica and pharmacologically characterized these channels. Our studies show that MdHCLB channels are more sensitive to HA and other agonists when compared with MdHCLA channels. GABA and biogenic amines act as agonists of MdHCLB channels and it is speculated that GABA may cross-react with MdHCLB channels endogenously in the nervous system. 5-HT and DA also act as competitive antagonists of HCLB channels. MdHCLB channels are also affected by antagonists but insensitive to known insecticides that target GABACls and GluCls.