Regulation Of Voltage-gated Sodium Currents By Endogenous Src Family Kinases In Cochlear Spiral Ganglion Neuons In Culture

Hearing loss is common in Otolaryngology. It is about10%of population suffer from different degrees of dearness in developed countries. The incidence of deafness in china is increasing also year by year because of these factors such as the abuse of antibiotics, noise pollution, population aging and genetic risk factors such as marriage. The prevention of hearing loss has become to be urgent to address in clinic. It is shown that the inhibition of tyrosine protein kinase, Src family kinase (SFKs), could prevent the cochlear damage from noise induced hearing loss. It is implied Src family may be involved in the pathological procedure of hearing loss.SFKs is a family of non-receptor kinase that includes nine members. SFKs is able to catalyse the transfer of phosphate from ATP to a tyrosine residue of specific cell protein targets, and modify the molecular structure and activity of substrate. The activity of SFKs is regulated by the phophosrylation of tyrosine527in C terminus and tyrosine416in activation loop of kinase domain. It is invoved in the regulation of neurons development, migration, survival and synaptic plasticity, and also mediated the excitotoxicity process in nervous system, which may be the pathological mechanism of epilepsy, pain, Alzheimer's disease and others neurodegenerative disease.Auditory information is converted as action potential in cochlear and then transmitted to the auditory central. Abnormal firing of auditory neurons can be lead to hearing loss. It is well known the voltage-gated sodium channel and potassium channels play critical roles in generation of action potentials. Some studies have indicated that the phosphorylated voltage-gated ion channels in auditory neurons would influence the firing of action potential. In addition, the voltage gated sodium and potassium channels are able to be regulated by SFKs in vivo. However, it is still unclear the effect of SFKs on sodium and potassium channels expressed in neurons, especially in auditory neurons. The cochlear spiral ganglion neurons (SGN) are primary auditory neurons, which interact with hair cells in peripheral and with auditory center in central. SGN is the origin of auditory action potential. Usually SGN is the target of excitotoxicity of noise or some ototoxic drugs. Dysfunction of voltage-gated sodium channels and potassium channels has proved to be induced hearing loss. Therefore, SGN cultured model in vivo was well established first by combination of cell primary culture technique and immunofluorescence staining in this study. The effect of endogenous SFKs on SGN voltage-gated sodium channel and potassium channel currents was investigated by whole cell configuration patch clamp technique. We also tested the regulation of I-V curve, activation curve and steady state inactivation curve of sodium channels and potassium channels by SFKs activator and inhibitor. To identify the role of Src in modification of action potential generated in SGN, we detected the influence of Src specific inhibitor Src40-58on the currents, macroscopic activation and inactivation, I-V curve, conductance, activation curve, steady state inactivation curve, recovery of INa on sodium and potassium channels. It will help us to illustrate the mechanism of cochlear impaired mediated by Src family, and provide a new guideline about deafness theorapy. This study is included three chapters:Chapter1:The regulation of voltage-gated sodium and potassium channels in spiral ganglion neurons by endogenous Src family kinaseObjective:To understand the mechanism of regulation of action potential generation by SFKs, the effect of voltage-gated sodium and potassium channels in spiral ganglion neurons (SGN) by SFKs inhibitors was detected.Methods:SGNs were dissected from cochlear tissue and plated into culture dishes. SGNs were identified by immunofluorescence staining. The location of SFKs in SGN was detected also. Inward and outward currents were indentified by TTX and Cs". To testify the effect on sodium channes and potassium channels in SGN by SFKs, the voltage-gated sodium and potassium channels currents were tested by whole cell configuration patch clamp technique after applied PP2, PP3, SU6656and vehicle. Subsequently, Na+in the extracellular solution was replaced with N-methy-D-Glucomine (NMDG) to block Na+currents. To investigate the regulation of Src kinase on voltage-gated potassium channel, potassium currents and Ⅰ-Ⅴ curve were detected when SFKs was applied.Results:(1) N-200selectively interacted with the membrane of SGN, and Hoechst3342selectively interacted with the nucleus of SGN. SFKs were detected in SGN by anti-SFKs antibody.(2) TTX and Cs+could block the inward currents and outward currents induced by depolarized pulse.(3) The sodium currents were significantly inhibited after applied10μM PP2or2μM SU6656. The currents is59.6±15.7%(n=9, P<0.05) and68.6±7.5%(n=5, P ＜0.05) compared with those recorded before SFKs inhibitors application. No significant change in the currents was found following application of PP3(10μM) or vehicle.(4) No change in potassium currents recorded at the same time was induced following application of PP2, PP3, SU6656or vehicle.(5) Potassium currents were recorded with Na+free extracellular solution in which Na+was replaced with NMDG. No change in potassium currents and I-V curve was detected following application of PP2, SU6656or vehicle.Conclusions:The inhibition of SGN voltage-gated sodium channel is induced by SFKs inhibitors. There is no change on potassium currents after inhibited SFKs. Chapter2:The regulation of kinetic of voltage-gated sodium channels in spiral ganglion neurons by endogenous Src family kinaseObjective:To investigate the mechanism of regulation on kinetic properties of voltage-gated sodium channels in SGN by SFKs.Methods:K+in the solutions was replaced with Cs+to block potassium currents. PP2. PP3. SU6656were diluted into extracellular solution, and Src activated peptide EPQ(pY)EEIPIA and control peptide EPQYEEIPIA were diluted into intracellular solution. The sodium currents, macroscopic activation and inactivation. I-V curve, conductance, activation curve, steady state curve and recovery of inactivation were tested following applied SFKs inhibitors or activators. Results:(1) The sodium currents were significantly reduced when applied10μM PP2or2μM SU6656. The currents were59.7±10%(n=7, P＜0.05) and62±8.8%(n=7, P＜0.05) respectively, when compared with those recorded before SFKs inhibitors application. No significant change in the sodium currents was found following application of10μM PP3or vehicle.(2) PP2and SU6656did not affect the macroscopic activation and inactivation.(3) Application of PP2or SU6656significantly reduced peak Na+current density in I-V curve without changing reversal potential. PP3or vehicle application did not produce such changes.(4) PP2reduced the conductance of sodium channel in SGN (control:6.7±1nS; PP2:5.2±0.7nS, n=7, P＜0.05). SU6656also reduced the conductance of sodium channel (control:11±2.4nS; SU6656:7.9±1.9nS, n=7, P＜0.05). PP3did not change the the conductance of sodium channel.(5) No change in activation curve of sodium channel was induced following application of PP2, PP3and SU6656.(6) The steady state inactivation curve was shifted to the left after PP2application (control:V1/2=-69.4±0.8mV, k=9.5±0.4; PP2:V1/2=-77.2±0.5mV, k=9.6±0.4, n=11,P＜0.05). Similarly, the steady state inactivation curve was also shifted to the left after application of SU6656(control:V1/2=-64.6±0.3mV, k=7.1±0.3; SU6656:V1/2=-70.1±0.4mV, k=7.7±0.4, n=9, P＜0.05). No significant change in the steady state inactivation curve was found following application of PP3.(7) The time constant of recovery of sodium channel was reduced by PP2(control:7.8±4.5ms; PP2:17.4±5.9ms; n=6, P＜0.05). The time constant of recovery of sodium channel was also reduced by SU6656(control:5±2.8ms; SU6656:11.2=2.2ms, n=6, P＜0.05). PP3did not affect the time constant of recovery. (8) The sodium currents were significantly potentiated when applied EPQ(pY)EEIPIA (1mM). The currents is131.3±5.2%(n=12, P＜0.05) comparing with the currents recorded immediately after the breakthrough. No significant change in the sodium currents was found following application of EPQYEEIPIA (1mM) or control pipette solution.(9) EPQ(pY)EEIPIA did not influence the macroscopic activation and inactivation.(10) Application of EPQ(pY)EEIPIA significantly increased peak Na+current density in I-V curve. EPQ(pY)EEIPIA did not change reversal potential. EPQYEEIPIA did not produce such changes.(11) EPQ(pY)EEIPIA inceased the conductance of sodium channel in SGN (control:8.2±1.3nS; EPQ(pY)EEIPIA:9.7±1.4nS, n=9, P＜0.05). EPQYEEIPIA did not produce such changes.(12) The activation curve was shifted to the left after EPQ(pY)EEIPIA application (control:V1/2:-36.4±0.3mV, k=3.5±0.3; EPQ(pY)EEIPIA:V1/2=-40.2±0.4mV, k=3.4±0.4, n=7, P＜0.05). No significant change in the activation curve was found following application of EPQYEEIPIA.(13) No change in activation curve of sodium channel was induced following application of either EPQ(pY)EEIPIA or EPQYEEIPIA.(14) The time constant of recovery of sodium channel was reduced by EPQ(pY)EEIPIA (control:5.6±2.5ms; EPQ(pY)EEIPIA:6.1±2.8ms, n=10, P＞0.05). EPQYEEIPIA did not produce such changes.Conclusions:Src family kinase regulates the voltage-gated sodium channels activtity in SGN. The conductance, activation and the steady-state inactivation of sodium channels are likely subjects to SFKs regulations. The effect on activation and inactivation of sodium channels is depended on the enzyme activity. Chapter3:The effect of Src on voltage-gated sodium channels in spiral ganglion neuronsObjective:To testify the regulation on voltage-gated sodium channel in spiral ganglion neurons by Src.Methods:Src40-58was diluted into intracellular solution. The sodium currents, macroscopic activation and inactivation, I-V curve, conductance, activation curve, steady state inactivation curve and recovery of INa were detected by whole cell configuration patch clamp technology. The effect of Src40-58on sodium currents in SGN was tested followed PP2pre-treated.Results:(1) The sodium currents were significantly reduced when applied Src40-58(0.3mg/ml). The currents were73.8±3.5%(n=10, P＜0.05) compared with those recorded before Src40-58application. Src40-58also reduced peak Na+current density in I-V curve without changing reversal potential.(2) Src40-58did not affect the macroscopic activation and inactivation.(3) Src40-58reduced the conductance of sodium channel in SGN (control:12±2.5nS:Src40-58:10±3nS. n=7. P＜0.05).(4) The steady state inactivation curve was shifted to the left following application of Src40-58(control:V1/2=-63.4±0.4mV, k=6.5±0.4; Src40-58:V1/2=-67.9±0.5mV,k=7.4±0.4, n=9, P＜0.05). Moreover, the activation curve was shifted to the left following application of Src40-58(control:V1/2=-38.5±0.4mV, k=3.8±0.4; Src40-58:V1/2=-44.3±0.6mV,k=3.2±0.5, n=7, P＜0.05).(5) The time constant of recovery of sodium channel was reduced by Src40-58(control:1.1±0.5ms; Src40-58:1.7±0.7ms, n=6, P＜0.05).(6) The inhibiton of Src40-58on the amplitude of sodium channel in SGN could be prevented in SGN bathed with solution with PP2(96.5±11.7%, n=10, P＞0.05). In this case, No change on peak Na+current density in I-V curve in SGN was found either.(7) The reduction on the conductance of sodium channel in SGN by Src40-58was prevented by PP2pre-treatment (control:8±0.9nS; Src40-58:7.5±1.2nS, n=7, P＞0.05).(8) The shift on steady state inactivation curve induced by Src40-58in SGN was prevented following PP2pre-treatment (control:V1/2=-71.2±0.4mV, k=6.7±0.4; Src40-58:V1/2=-73.3±0.4mV, k=7.1±0.3, n=8, P＞0.05). However, following application of Src40-58, the activation curve was still shifted to the left in SGN when was pre-treated by PP2(control:W1/2=-33.8±0.5mV, k=3.7±0.7; Src40-58: V1/2=-39.9±1mV, k=4.8±0.8, n=9, P＜0.05).(9) Following application of Src40-58, no change on the time constant of recovery of sodium channel was found in SGN bathed with PP2(control:7±4.6ms:Src40-58:7.4±4.6ms; n=6, P＞0.05).Conclusions:Src is involved in the regulation of sodium channels in SGN. The effect of Src on sodium channels in SGN is similar as that of SFKs...