Role of ion channels from the Cys-loop family in autonomic function

5-羟色胺能3型受体（5 HT 3Rs）是配体门控离子通道的Cys环家族的成员，它们与烟碱ACh、GABAA和甘氨酸受体共享。该家族的所有成员在神经系统中广泛表达，它们主要参与快速突触传递。在外周，nAChR的激活驱动交感神经节和副交感神经节处的快速突触传递;而迷走感觉神经末梢上的5 HT 3R的激活影响呼吸、循环、呕吐和伤害感受。尽管5 HT 3R在自主神经元中功能性表达，但其功能尚未完全确定。在本研究计划中，我们将致力于以下研究目标：*1）5-HT-3受体与nAChRs之间的交叉抑制：我们将研究小鼠上级颈神经节（SCG）交感神经元中5-HT-3受体激活引起的乙酰胆碱诱发电流的交叉抑制机制。由于nAChR驱动交感神经节中的自主神经功能，因此这种调节机制对于更好地理解5 HT 3R对自主神经功能的作用变得至关重要。我们将通过共刺激5 HT 3R和nAChR的膜片钳技术来检查串扰，从而产生组合电流。*2）M3-M4细胞内接头在串扰抑制中介导的受体相互作用中的作用：我们提出利用先前公开的嵌合-5HT3R，其中M3-M4接头已被原核同源物取代。该受体保留其功能，但不与伴侣蛋白相互作用，将用于测试5 HT 3Rs和nAChRs之间可能的相互作用。我们将在非洲爪蟾卵母细胞中共表达这些受体与nAChRs，以测试它们的串扰机制。 *3）5 HT 3R在自主神经功能中的作用：为了测试5 HT 3R对自主神经系统的正常功能的相关性，我们将研究来自野生型（WT）和5 HT 3A敲除（KO）小鼠的完整自主神经节中的快速突触传递。由于5 HT 3A KO小鼠不表达功能性5 HT 3R，因此它们允许在不存在由5 HT 3R介导的调节作用的情况下研究自主功能的参数。* 我们预计这项研究将揭示5 HT 3R在自主神经功能中通过调制或“调谐”烟碱突触传递的新生理作用。

Serotonergic type 3 receptors (5HT3Rs) are members of the Cys-loop family of ligand-gated ion channels, which they share with nicotinic ACh, GABAA and glycine receptors. All members of this family are widely expressed in the nervous system where they mostly participate in fast synaptic transmission. In the periphery, activation of nAChRs drive fast synaptic transmission at sympathetic and parasympathetic ganglia; while activation of 5HT3Rs on vagal sensory nerve endings affect respiration, circulation, emesis and nociception. Although 5HT3Rs are functionally expressed in autonomic neurons their function has not been yet fully determined. In the current grant proposal we will concentrate in the following research aims:***1) Cross-talk inhibition between 5HT3Rs and nAChRs: We will investigate cross-inhibitory mechanisms on ACh-evoked currents elicited by 5HT3R activation in cultures sympathetic neurons from the superior cervical ganglion (SCG) of mice. Because nAChRs drive autonomic function in sympathetic ganglia, this modulatory mechanisms becomes of crucial interest to better understand the role of 5HT3Rs on autonomic function. We will examine cross-talk by the patch-clamp technique by co-stimulating 5HT3Rs and nAChRs, thus generating a combined current. ***2) Role of the M3-M4 intracellular linker in mediated receptor interaction in cross-talk inhibition: We propose to take advantage of a previously published chimeric-5HT3R which M3-M4 linker has been replaced by a prokaryotic homologue. This receptor, which conserve it s function but does not interact with partner proteins, will be used to test the possible interaction between 5HT3Rs and nAChRs. We will co-express these receptors with nAChRs in Xenopus oocytes to test their cross-talk mechanisms.    ***3) 5HT3R role in autonomic function: To test the relevance of 5HT3Rs on the normal function of the autonomic nervous system we will study fast synaptic transmission in intact autonomic ganglia from wild type (WT) and 5HT3A knock out (KO) mice. Since 5HT3A KO mice do not express functional 5HT3Rs they allow to study parameters of autonomic function in the absence of the modulatory effects mediated by 5HT3Rs. ***We anticipate this research will reveal a new physiological role for 5HT3R in autonomic function by modulation or "tuning" nicotinic synaptic transmission.***********