Molecular Characteristics And Physiological Function Of Sodium Channels In Developing Cochlea

Spontaneous action potential(sAP)is a common characteristic of sensory systems during the early development stage.It plays an important role in the process of development and mature of neural circuits which in charge of sensory information processing.Before onset of hearing,the spontaneous action potential originated from cochlear hair cells,induces burst-discharge activity of spiral ganglion,and then transmits to auditory center accurately.Accumulation data hint that sAP firing in IHCs is found to be associated with a inward calcium current carried by the Cav1.3,a slow delayed rectifier potassium outward current and a small conductance Ca2+-activated K+current SK2.However,voltage-gated sodium channels(VGSCs)known as the major contributors for the rising phase of action potential are less concerned.The frontier academic questions such as molecular characteristics,dynamic expression pattern,biophysical properties and physiological function need to be approached.This study focuses on:1)The a subunits of all known VGSC subtypes(Nav1.1?Nav1.9)were detected at mRNA level in developing cochlear sensory epithelia.Then,the a subunits of nine VGSC subtypes were cloned and analysed from developing cochlear sensory epithelia.Sequence analysis showed that sequences of mNav1.1 and mNav1.4 are same to variants which expressed in other mice tissues.The other seven VGSCs showed some unique mutations scatteredly distributed in functional areas,and then produced distinct subtype variants,as a result of undergoing a series of transcriptional regulation such as alternative splicing and RN A editing.For this,they were named as CbmNav1.x.2)At the same time,the nine subtypes were also detected in IHCs and OHCs at protein level,the combinatorial expression pattern of VGSC subtypes were the same.On the other hand,the differential gene expression profiles of apical-basal sensory epithelia showed that,the mRNA expression level of Nav1.5 a subunit was higher in apical sensory epithelia,Nav1.1 a subunit and ?4 subunit were tender to gather in basal sensory epithelia.Furthermore,data obtained by single-cell qPCR presented that the expression level of nine subtypes in IHCs and OHCs were time-and position-dependent.In IHCs the expression level of Nav1.7 and Nav1.9 was higher compared with other subtypes,showing the similar dynamic change trend and peak appeared at postnatal 8 day,and the expression quantity of the remaining seven VGSC subtypes were relatively low but showed unique trends.In OHCs,expression quantity of other eight sodium channels was extremely low compared to Nav1.7,which suggested that Nav1.7 might be the dominant subtype contributing to sodium current.These mentioned above strongly implied that the alternative combination approach and time/position-dependent expression pattern of VGSC subtypes might be important physiological basis for dynamic regulation of sAP in developing HCs.3)Functional identification showed that the currents of CbmNavl.7a and CbmNav1.7b cannot be detected,an amino acid residue replacement(C(934)R)located the extracellular linker between segment 5 and 6 of domain ? silences CbmNav1.7.In addition,the lacking of part of segment 3 and segment 4 in Domain? of CbmNav1.7b produces a non-functional truncated channel protein.It was speculated that the residue replacement and truncated protein might be endogenous regulation mechanism of HCs.Then,it is identified that the sodium current of HCs might be contributed by functional CbmNav1.7a(C934),and it gating properties were investigation.In comparision with DRG-type Nav1.7,the function of CbmNav1.7a(C934)was erratic.The residue replacements and deficiencies distributed in four functional areas could modify channel dynamics parameters with separate or combinational way,and then make CbmNav1.7a(C934)more suitable for activation at subthreshold potential and shaping sAP.4)The typical voltage-dependent sodium currents were recorded in developing inner hair cells(IHCs)and outer hair cells(OHCs).Current clamp recording showed that the sAP cannot be detected in OHCs,the current injections(70 pA/90 pA,400 ms)only induce single spike firing,whereas the continuous spontaneous spike activity could be detected in IHCs.The difference between two might attributed to the lacking of SK2 channel in OHCs which mediating sAP firing.The sAP in IHCs could be eliminated by incubation of Ca2+-free extracellular fluid,which hinted that the sAP firing is Ca2+ current-dependent.500 nM TTX could reduce the firing frequency,but without effect on amplitude of sAP.Analyses of AP space-time parameters revealed that 500 nM TTX could reduce the maximum rate of rise of the upstroke of the action potential and the rate of subthreshold depolarization.Moreover,the width of the action potential at the subthreshold(20%of the spike amplitude)was increased by 500 nM TTX.In other words,The Na+ currents shorten the time to reach the action-potential threshold and then increase the firing frequency.These data clearly hint that,in vivo the sodium current of IHCs might be in hearing maturation by regulating the frequency of action potential and neurotransmitter release.