Membrane Dynamics Of Piezo2 Mechano-gated Channelsin DRG Neurons And Its Role In The Inflammatory Pain

In 2010,Coste et al.demonstrated that Piezo mechano-gated ion channels,including Piezo1 and Piezo2,were identified as the long-sought-after molecular carrier of mechanically-activated current in mammalian cells.Piezo1 and Piezo2,are expressed in a variety of tissues including the bladder,colon and lung,but Piezo2 is dominantly expressed in the Dorsal root ganglion?DRG?neurons.Piezo2 channels mediates the rapidly adapting mechanically-activated?RA?currents,which inactivation time constant?the?value?is less than 10 ms.Piezo channels,as the only recognized mechano-gated ion channels in mammals,its structure,biophysical properties,functional regulations and its physiological and pathophysiological significance have been revealed more and more.Conditional knockout of the Piezo2 channel in the mouse DRG sensory neurons significantly impaired their low-threshold tactile sensations.Loss-of-function mutations in Piezo2 channel could lead to the loss of low-threshold tactile sensation in human glabrous skin,and the decreased ability of two-point touch discrimination.Inhibition or knockout of the Piezo2channel in DRG neurons could eliminate the mechanical allodynia?MA?in animals with inflammatory or neuropathicconditions.Thus,Piezo2 mediated the low-threshold touch sensation in physiological condition but mediated low-threshold touch-induced MA in pathophysiological conditions.Although it has been confirmed that Piezo2 is the molecular basis for mediating MA,its underlying molecular mechanism remains unknown.Our previous study demonstrated that intracellular hyper-osmolarity or extracellular hypo-osmolarity-induced DRG neurons swelling,which significantly increased the static plasma membrane tension?SPMT?,as well as significantly potentiated the Piezo2/RA currents in DRG neurons.Plantar injection of hypotonic solution into the hindpaw could elicit MA in animals.Thus,changes in cell SPMT are important molecular basis for the regulation of Piezo2 channels.Using the non-stationary fluctuation analysis to assess the channel numbers and unitary current size when the DRG neurons underwent osmotic swelling to increase SPMT.The channel numbers in plasma membrane increased for about 4-fold,while the unitary current size only showed an increasing trend without significant change.We used Western blot to detect the total expression of Piezo2 channels in DRG neurons after extracellular hypotonic solution induced cell swelling,there was no significant difference between isotonic and hypotonic conditions.Taken togather,these results indicated that Piezo2 channels may traffic to plasma membrane from some“hidden places”in the intracellular side of DRG neurons,and then,increased the channel numbers of Piezo2 in plasma membrane,significantly potentiated the Piezo2/RA current,which mediated the MA produced in multiple pathological conditions.The question is,where are these Piezo2channels hiding?Caveolae and Clathrin-coated pits?CCP?,both are typical cell membrane invagination structures,which contained various cargos,signal proteins or others,are the main internalization?endocytosis?pathways from plasma membrane to the cytosol.Caveolin-1 and Clathrin are the assembly promoter proteins of the internaliztion of Caveolae and CCP,respectively.On the other hand,Caveolae and CCP function as the membrane reservoir,could terminate the internalization,traffic and flatten to supplement the cell membrane in responding to the increased plasma membrane tension.Signal molecules hidden in these invagination structures also traffic to the cell plasma membrane.In summary,we hypothesize that a quantity of Piezo2 channels may distribute in the cell membrane invagination structures such as Caveolae and/or CCP in DRG neurons.The plasma membrane Piezo2 channels elicit Piezo2/RA currents in responding to mechanical stimulation to mediate low-threshold touch.Whereas,the Piezo2 channels‘hiding'in the invagination structures that donot elicit Piezo2/RA currents since they couldn't receive the effective mechanical stimulation.Pathological conditions such as cell swelling,inflammation or nerve damage may significantly increase the number of Piezo2 channels in the cell membrane,enhance Piezo2/RA currents through the invagination structures translocating and flattening to the cellular membrane when they are subjected to the increased SPMT,which results in MA of chronic pain.This project will be divided into two sections to study the membrane dynamics of Piezo2 mechano-gated channels in DRG neurons and its role in the inflammatory pain.Part one:Study on membrane dynamics of Piezo2 channels in DRG neuronsObjective:To study the distribution of Piezo2 channels in Caveolae and/or CCP;to study the membrane dynamics?Internalization-Membrane trafficking?of Piezo2 channels following caveolae and/or CCP pathways.Methods:?1?Caveolin-1 and Clathrin were used as markers for Caveolae and CCP,respectively.Using laser scanning confocal microscopy?LSCM?,total internal reflection laser confocal microscopy?TIRFM?,Stochastic Optical Reconstruction Microscopy?STORM?and fluorescence resonance energy transfer?FRET?techniques to study the colocalization of Piezo2 channels and Caveolin-1 or Clathrin,and the interaction between Piezo2 and Caveolin-1 or Clathrin.?2?Using LSCM,TIRF,STORM and Western Blot?WB?,to study the membrane dynamics?Internalization-Membrane trafficking?of Piezo2channels and the underlying mechanism.The expression changes of Piezo2channel is also studied in DRG neurons.?3?The effect of membrane dynamics of Piezo2 channel on Piezo2/RA current was studied by patch clamp electrophysiological technique.Results:1.It is well known that KCNQ2 and KCNQ3 co-localized and firmed M-type K⁺channel in DRG neurons,while KCNQ2 and KCNQ4 did not do.So,the colocalization of KCNQ2 and KCNQ3?Q2+Q3?was used as a positive control and the location of KCNQ2 and KCNQ4?Q2+Q4?was used as a negative control,to verify the reliability of the STORM system.The experimental results show that KCNQ2 and KCNQ3 have obvious co-localization,but no co-localization between KCNQ2 and KCNQ4,which indicate that the STORM system in our laboratory is running well.2.STORM,LSCM/TIRF and FRET experiments demonstrated the obvious co-localization between Piezo2 and Clathrin,or Caveolin-1,respectively.The FRET and the STORM?distance-peak between two milecules,ranged from 20 nm to 100 nm?experiments indicate that Piezo2channel may interact with Clathrin or Caveolin-1,respectively.3.Two lines of factors were used to study the membrane dynamics?Internalization-Mmebrane trafficking?of Piezo2 channel followed Caveolae and/or CCP.One is internalization promoting factos,Cytochalasin D?CD?.After incubation of DRG neurons by CD,Clathrin,Caveolin-1 and Piezo2significantly internalized,the cytoplasmic distribution increased,and the distribution in cell membrane was significantly reduced.The other line factors,such as Dynasore,Hypo-osmolarity and MBCD,which inhibit the internalization and promote the membrane trafficking of signal proteins through Caveolae and/or CCP.After incubation with Dynasore,Hypo-osmolarity or MBCD,Clathrin and Caveolin-1,as well as Piezo2 were significantly translocated to the cell membrane,and their cytoplasmic distribution was significantly decreased.CPZ?chlorpromazine?,a selective inhibitor of CCP-induced internalization,incubating DRG neurons can lead to a significant membrane trafficking of Clathrin and Piezo2 in DRG neurons.The WB experiments demonstrated that no significant changes were shown in the expression of Piezo2 channel in DRG neurons,when the cells underwent the treatment by the above factors.4.Patch clamp electrophysiological experiment indicated that Dynasore significantly increased the Piezo2/RA current in DRG neurons.Conclusion:1.KCNQ2 and KCNQ3,as a positive control,have obvious co-localization,and as a negative control,there is no colocalization between KCNQ2 and KCNQ4.The reliability of STORM system we used is good.2.LSCM/TIRF,FRET and STORM data indicate that Piezo2 is co-localized with Clathrin or Caveolin-1 and may exist interactions between Piezo2 and these assembly proteins in DRG neurons.The membrane dynamics?Internalization-Membrane trafficking?of Piezo2 channels following caveolae and CCP pathways were confirmed in DRG neurons.3.Electrophysiological data indicate that the alteration in membrane dynamics of Piezo2 mechano-gated channel accounts for the changes of Piezo2/RA current.Part two:The alteration in membrane dynamics of Piezo2 channel mediated the MA in CFA inflammatory model animalsObjective:Incubating DRG neurons with NGF or inflammatory soup?mixture of PGE₂,5-HT,BK,His?to study the membrane dyniamics of Piezo2channel;to study the alteration in membrane dynamics of Piezo2 channel in mediating MA in CFA chronic inflammatory animal model.Methods:?1?DRG neurons were incubated with NGF or inflammatory soup to observe the membrane dynamics of Piezo2,Clathrin and Caveolin-1with TIRF and STORM.?2?CFA was injected into the hindpaw to induce animal model of inflammation.The behavior of the animals was measured by von Frey Filament and thermal pain meter.The edema volume of the hindpaw with CFA injection was measured.?3?Using LSCM/TIRF and STORM techniques to observe the membrane dynamics of Piezo2 channel in DRG neurons in of CFA inflammatory rats.?4?Using patch clamp electrophysiological technique to record the Piezo2/RA currents in DRG neurons incubated with NGF or inflammatory soup;to record the Piezo2/RA currents in DRG neurons of CFA inflammation model rats.Results:1.LSCM/TIRF and STORM data showed that incubation of DRG neurons with NGF or inflammatory soup could significantly induce membrane trafficking of Piezo2 channels,Clathrin and Caveolin-1.2.Intraplantar injection of CFA into the hindpaws in both sides to induce CFA chronic inflammatory model.N.S.injection was used as negative control.The withdrawal threshold of mechanical stimulation and the withdrawal latency of thermal stimulation were measured,respectively.The results showed that the mechanical withdrawal thresholds were signifcantly reduced in CFA animals;the thermal withdrawal thresholds of CFA model animals were significantly lower than that of the N.S.group.Moreover,the degree of swollen hindpaws in CFA inflammatory group was significantly higher than that of the control group.3.LSCM/TIRF and STORM results showed that the significant membrane trafficking of Piezo2 channel,Clathrin and Caveolin-1 were observed in DRG neurons inCFA inflammatory model animals.4.Patch clamp recordings showed a significant potentiation of Piezo2/RA current in DRG neurons incubated with NGF or inflammatory soup.Conclusion:1.Incubating DRG neurons with NGF or inflammatory soup,significantly promote the the membrane trafficking of Piezo2 channels,Clathrin and Caveolin-1;potentiate the Piezo2/RA currents.2.The mechanical or thermal hypersensitivity were induced in CFA inflammatory model rats.3.CFA inflammation induced a significant membrane trafficking of Piezo2 channels in DRG neurons.