The Dual Regulation Mechanism Of Potassium Channel Auxiliary Subunit Kv?1 On TRPV1 Channel Function

TRPV1 channel is a non-selective cation channel,which is widely expressed in sensory neurons and non-sensory neuron cells.As a multisensory nociceptor,TRPV1 channel can respond to a variety of physical and chemical stimuli to participate in the maintenance of body homeostasis.The perception of pain is the research hotspot of TRPV1.The body is often accompanied by inflammatory hyperalgesia,mechanical hyperalgesia and thermal hyperalgesia in the occurrence of diseases such as tissue damage,inflammation and cancer.The production of these pain sensations is often related to the abnormal function of the TRPV1 channel.Therefore,it has important physiological and pathological significance to explore the function of TRPV1.Regarding the regulation of TRPV1 function,in addition to using chemical reagents such as agonists or inhibitors to directly regulate the gating of the channel,studies have found that a variety of ligand proteins can also regulate the function of the channel by interacting with the TRPV1 channel.Such as forming heteromultimers with other TRP channel members;post-translational modifications caused by interaction with protein kinases,phosphatases and SUMO-related enzymes,and forming signal complexes with specific protein molecules to regulate the expression and function of the channel.In view of the fact that the signal complex is a state of protein interaction under physiological conditions,this regulation method has more physiological significance for the regulation of TRPV1 channels.To identify new proteins that form signal complexes with the TRPV1 channel,a yeast two-hybrid experiment was first performed to screen the yeast two-hybrid c DNA library prepared from the mouse dorsal root ganglion.After multiple auxotrophic screening and preliminary bioinformatics analysis,we finally obtained 18 potential candidate library proteins that interact with the N-terminus of the TRPV1 channel,one of which is the voltage-gated potassium channel auxiliary subunit Kv?1.Since the research on the Kv?1 subunit is mainly focused on exploring its regulation of the function of potassium ion channels,there are few studies on the regulation of this subunit on other ion channels,and the yeast two-hybrid screening results show that the Kv?1 subunit has a strong interaction with the TRPV1 channel.Therefore,this thesis explores the functional regulation of the Kv?1 subunit on TRPV1 channels through molecular biology,biochemistry and electrophysiological patch clamp experiments,and the following results were obtained:(1)Through yeast rotation,GST pull-down and immunoprecipitation experiments,it was found that Kv?1 subunit and TRPV1 channel directly interacted with each other both in exogenously overexpressed HEK 293 T cells and endogenously expressed DRG neurons.(2)The expression of Kv?1 subunit can significantly increase the current density of TRPV1 channels without affecting the sensitivity of TRPV1 channels to voltage,capsaicin and acidic p H,and this subunit also increases the temperature activation threshold of TRPV1 channels.(3)Exogenous overexpression of Kv?1 subunit can significantly increase the expression of TRPV1 channel on the cell membrane without changing the total amount of channel protein.(4)The Kv?1 subunit interacts with the two N-terminal domains of the TRPV1 channel to play a dual regulatory role in the function of the TRPV1 channel.The Kv?1subunit interacts with the membrane distal domain of the TRPV1 channel to increase the amount of expression on the membrane,while the interaction with the membrane proximal domain increases the temperature activation threshold of the TRPV1 channel by 3 ?.In summary,this paper has discovered a new protein molecule Kv?1 subunit that forms a signal complex with TRPV1 channel,and revealed the specific mechanism of the functional regulation of TRPV1 channel by this subunit.In addition,we also found that the Kv?1 subunit also interacts with TRPV2,TRPA1 and other TRP channels.This result provides a good reference for the follow-up exploration of Kv?regulation of other TRP channels in sensory neurons.