ASICs: Subunit Assembly And Their Roles In Dendritic Development In Hippocampal Neuron

ASICs are cationic channels activated by extracellular protons,widely expressed in nervous system.They play multiple roles in both physiological and pathological conditions,such as mechanical perception,taste,vision,nociception,synaptic plasticity,memory,ischemia and seizure etc.ASICs belong to the ENaC/DEG (epithelial amiloride-sensitive Na+ channel and degenerin)family of ion channels. They share the same overall structure with other members of this family:two hydrophobic transmembrane regions(TM1 and TM2),a large extracellular loop comprising many conserved cysteines,and the C and N termini facing the intracellular space.So far,six ASIC subunits encoded by four genes have been cloned, ASIC1a and its splice variant ASIC1b,ASIC2a and its splice variant ASIC2b,ASIC3 and ASIC4.In the CNS,neurons mainly express ASIC1a,2a and 2b subunits,while ASIC1a is the predominant functional subunit.Homomeric ASIC1a channels mediate a fast and transient inward current,conducting Na⁺ and Ca²⁺ions.The pH for half maximal activation(pH_0.5)in homomeric ASIC1a channels was 6.2.Homomeric ASIC2a channels have a low sensitivity to protons with a pH_0.5of～4.4.ASIC2b subunits do not form functional homomeric channels,but they may associate with other subunits to form heteromeric ASICs with distinct properties.Functional ASICs are usually believed to be tetrameric assemblies of homomeric or heteromeric subunits.However,recent Gouaux and colleagues' crystal structure shows that there are three subunits in ASIC1 homomeric channel.Recent studies have shown that ASICs play an important role in the CNS, contributing to synaptic plasticity,learning and memory and axonal degeneration. Welsh lab(2002)generated ASIC1 knockout mice and found that loss of ASIC1 impaired hippocampal-dependent LTP,spatial learning and eye-blink conditioning. Then this group also found that ASIC1 knockout mice showed a reduced fear response to both cued and context fear conditioning.Conversely,overexpressing ASIC1 in the amygdala and elsewhere in the brain enhanced context fear conditioning. A recent study using organotypic hippocampal slices shows that ASIC1a,localized in dendritic spines,can affect the density of spines.Decreasing ASIC1a reduced the number of spines,whereas overexpressing ASIC1a had the opposite effect.The mechanism is through influencing intracellular Ca²⁺concentration and CaMKII phosphorylation.Friese et al.(2007)found that compared to wild-type mice,ASIC1 knock out mice showed both a markedly reduced clinical deficit and reduced axonal degeneration after induction of experimental autoimmune encephalomyelitis(EAE), which suggested that ASIC1 contributes to axonal degeneration in autoimmune inflammation of the central nervous system.In this dissertation,we studied the assembly of ASICs in living cells and the role of ASICs in dendritic development in hippocampal neuron.1.Fluorescence resonance energy transfer analysis of subunit assembly of the ASIC channelHomo-and heteromeric ASIC complexes have been verified by classical methods such as co-immunoprecipitation,or functional assays applying electrophysiology.However,the exact subunit assembly and stoichiometry of heteromeric ASIC channels in living cells are largely unknown.In the present study, we have used a biophysical approach based on Fluorescence Resonance Energy Transfer(FRET)to address these questions directly in living cells.For FRET measurement is non-invasive,it allows analysis of the interaction between proteins in living cells.In addition,FRET has high spatial resolution.It can serve as "a molecular ruler" and has been successfully applied in the investigations of stoichiometry and assembly of many types of receptors and channels.We fused ASICs subunits with CFP and YFP,and transfected these constructs into CHO cells. We get the FR value through three-cube FRET imaging,then FRET efficiency between neighboring subunits was estimated according to the tetramer or trimer FRET model.Our results showed that when either tetrarner or trimer FRET model was used,co-expression of the same ASIC protein fused with CFP and YFP produced significant FRET signals,consistent with the homomeric assembly of ASIC channels. When two different ASIC proteins fused with CFP and YFP,respectively,were co-expressed in the cell,significant FRET signals were also produced,suggesting the occurrence of hetermeric assembly of ASIC proteins.In addition,we studied the stoichiometry of hetermeric ASIC complex.Our results suggested that the channel contains two of each subunit and the stoichiometry is apt to 2:2 if hetermeric ASIC channel is a tetramer,while the assembly of ASICs is random,the stoichiometry is not fixed(2:1 and 1:2 coexist)if hetermeric ASIC channel is a trimer.This study provided interesting new data supporting the occurrence of the homomeric and heteromeric assembly of ASICs.The existence of many isoforms and subunits creates functional heterogeneity of ASICs,which to some extent determines the variable and complicated biological properties that ASICs have in the brain and peripheral sensory neurons. 2.The role of ASIC channel in dendritic development in hippocampai neuronRegulated growth and arborization of dendritic processes are critical to the formation of functional neuronal networks.However,the role of ASICs in dendritic development is still elusive.In the present study we transfected F-GFP into cultured hippocampal neurons at 5 days in vitro(DIV 5).Then ASICs antagonists were applied to the neuronal medium to inhibit the function of ASICs.Dendritic growth and arborization of cultured hippoeampal neuron were observed in DIV8 and DIV14. We attempted to determine whether and how ASICs regulate dendritic development. Our results showed that the total dendrite branch length and arborization of hippocampal neuron were not affected by the inhibition of ASICs for a short time, while the total dendrite branch length was reduced and dendrite development was impaired if ASICs were inhibited for a long time.It suggested that ASICs play a role in the dendrite development,which may help understand how ASICs affect the formation of neuronal networks and participate in higher brain fuctions such as learning and memory.