In vertebrates and invertebrates, OTOP channels expressed in the gustatory system sense acids and function as sour taste receptors (OTOP1 for vertebrates OTOPL1 for Drosophila) ( Teng et al., 2019 Zhang et al., 2019 Ganguly et al., 2021 Mi et al., 2021). OTOP proton channels are expressed throughout the body, where they play diverse and still poorly understood roles in pH sensing and homeostasis. OTOP1 is a member of a family of proteins ( Hughes et al., 2008 Hurle et al., 2011), which includes, within vertebrates, two other members, OTOP2 and OTOP3, that also function as proton channels ( Tu et al., 2018). Zn 2+ is a potent inhibitor of proton transport molecules including the voltage-gated proton channel Hv1 and the proton-selective ion channel OTOP1 ( Decoursey, 2003 Ramsey et al., 2006 Bushman et al., 2015 Tu et al., 2018 Teng et al., 2019). Zn 2+ binds to proteins with high affinity and specificity and regulates a wide range of cellular processes, including metabolism and gene expression ( Vallee and Falchuk, 1993). One of the most common modulators of channel activity is the trace metal zinc (Zn 2+), which can affect gating, permeation, or both ( Gilly and Armstrong, 1982 Chu et al., 2004 Noh et al., 2015 Peralta and Huidobro-Toro, 2016). More recently, toxins that target pain-sensing ASIC and TRPV1 channels have been used to probe the conformational states of these channels ( Bohlen et al., 2010 Baconguis et al., 2014). Similarly, gating modifiers have been used to probe structural rearrangements that accompany the opening of voltage-gated ion channels ( Swartz and MacKinnon, 1997 Sack and Aldrich, 2006 Catterall et al., 2007 Goldschen-Ohm and Chanda, 2014). For example, the discovery of the charged molecule TEA and the scorpion toxin charybdotoxin as a specific blocker of K + channels allowed for the early identification of residues lining the channel pore well before the channel structures were determined ( MacKinnon et al., 1990 Yellen et al., 1991 Banerjee et al., 2013). Pharmacological agents that can activate or inhibit ion channels have long been used as probes to describe the fundamental processes of channel gating and ion permeation ( Hille, 2001). Zn 2+ is an essential micronutrient and its activation of OTOP channels will undoubtedly have important physiological sequelae. These results establish the tm 11–12 and tm 5–6 linkers as part of the gating apparatus of OTOP channels and a target for drug discovery.
Kinetic modeling of the data is consistent with Zn 2+ stabilizing the opn2+en state of the channel, competing with H + for activation of the channels. Mutation to alanine of H531 and E535 within the tm 11–12 linker and H234 and E238 within the 5–6 linker reduced or eliminated activation of mOTOP3 by Zn 2+, indicating that these residues likely contribute to the Zn 2+ activating site. Swapping the extracellular tm 11–12 linker between mOTOP3 and mOTOP2 was sufficient to eliminate Zn 2+ activation of mOTOP3 and confer Zn 2+ activation on mOTOP2. In contrast, mOTOP2 currents are insensitive to activation by Zn 2+.
Zn 2+ pre-exposure increases the magnitude of mOTOP3 currents to a subsequent acid stimulus by as much as 10-fold. Here, we show that zinc (Zn 2+), as well as other transition metals including copper (Cu 2+), potently activates murine OTOP3 (mOTOP3). Importantly, the gating mechanisms of any of the OTOP channels are not well understood. In vertebrates there are three family members: OTOP1 is required for formation of otoconia in the vestibular system and it forms the receptor for sour taste, while the functions of OTOP2 and OTOP3 are not yet known. Otopetrin proteins (OTOPs) form proton-selective ion channels that are expressed in diverse cell types where they mediate detection of acids or regulation of pH.
0 kommentar(er)
