Abstract The Negative Modulation On N-type Calcium Channel By Sigma-1 Receptor And The Mechanism Of The Effect

Sigma receptor is a unique kind of receptor with chaperone activity.Due to it has no homology with any other mammalian proteins,and its special function in organisms compared with other receptors,now it is regarded as an orphan receptor.Sigma receptor family contains two subtypes: one is Sigma-1 receptor and the other one is sigma-2 receptor.Because we have not obtained the clone of sigma-2 receptor and the sequence of amino acids is still unclear till now,there is less study on sigma-2 receptor than on Sigma-1 receptor.Lots of researches have been focused on Sigma-1 receptors.Sigma-1 receptors mainly aggregate at the ER?Endoplasmic Reticulum?,and they are widely distributed at the membrane system,such as MAM?mitochondria-associated membrane?,plasma membrane and nuclear membrane.After the research on Sigma-1 receptor for nearly 40 years,especially after we obtained the clone of Sigma-1 recepor and the Sigma-1 receptor knock-out mouse,the structures and the functions of this receptor are more and more clear to us.Sigma-1 receptor contains 223 amino acids with a single protein strand,it weights about 26 k Da.In previous study,Sigma-1 receptor was regarded as a protein with two transmembrane domains,while both N-terminal and C-terminal are inside the cell membrane.However,in the year of 2016,the analysis of human Sigma-1 receptor crystal structure demonstrated a trimeric organization of Sigma-1 receptors.It contains only a single transmembrane domain for each promoter,and the domain includes a cupin-like ?-barrel with the ligand-binding site buried at its center,with N-terminal at the outside of the cell membrane and C-terminal at the inside of the plasma membrane.Sigma-1 receptors are very significant in regulating many physiology processes and diseases,such as mnemonic impairment,cancer,cardiac diseases,stroke,depression,AD and psychiatric disorders.Sigma-1 receptors expressed in the central nervous system are the most abundant among these organisms,and the functions are the most complex.By using the method of electrophysiology and behavioral pharmacology,studies have demonstrated that Sigma-1 receptors participated in many diseases and pathology processes in central nervous system,such as depression,anxiety,schizophrenia,learning and memory,addiction and pain.By analyzing the factors that could induce these diseases,we found that they are all related with the disorder of the neurotransmitter release and transduction.As previous reports,Sigma-1 receptors could decrease or potentiate the release of glutamatergic,dopaminergic,serotonergic,noradrenergic and cholinergic neurotransmitters,but the mechanism of the regulation is still a mysterious now.Activation of the N-type Ca²⁺ channels can induce the release of the neurotransmitters,and it can directly regulate the transduction and exocytosis of the synapses.In this way,N-type Ca²⁺ channels play an important role in regulating neurotransmitters.According to the advancements in researching Sigma-1 receptors,we learn that the Sigma-1 receptors can interact with many voltage-gated and ligand-gated ion channels?including potassium channels,sodium channls,acid-sensing ion channels and so on?,and induce many physiological effects through affecting the electrophysiological characterization of these ion channels.So,we speculate if Sigma-1 receptor could regulate the release and transduction of the neurotransmitter by affecting the N-type Ca²⁺ channels.However,there are no previous reports about the relationship between the Sigma-1 receptors and N-type Ca²⁺ channels.That is to say,we are not clear about that whether Sigma-1 receptors can control the neurotransmitter release through regulating N-type Ca²⁺ channels.To explore the influence on neurotransimitter releasing by Sigma-1 receptors,we select N-type Ca²⁺ channels as our breakthrough point in the present study.This experiment aims to discuss the Sigma-1 receptor's regulation on N-type Ca²⁺ channels.Regarding the ChIs are the only neurons that could synthesize and release Ach,in the present study,we demonstrated the expression of the Sigma-1 receptors in ChIs for the first time using sc RT-PCR and immunofluorescence.And by sc RT-PCR,we proved the N-type Ca²⁺ channel was the prominent subtype expressing in ChIs.These results illustrated us the ChIs are the ideal model for our experiments.The whole cell clamp patch on brain slice showed the Sigma-1 receptors inhibited N-type Ca²⁺ channels in ChIs.Considering the advantages of Xenopus oocytes in researching ion channels,we used oocytes for the further study on N-type Ca²⁺ channels.The results indicated us the Sigma-1 receptors could inhibit the N-type Ca²⁺ channels in a ligand-independent and ligand-dependent manner.To verify the speculation on the mechanism,FRET and co-immunoprecipitation were performed and the results indicated the direct interaction between the Sigma-1 receptors and the N-type Ca²⁺ channels in HEK-293 T cells.In brief,we used the whole cell clamp patch on brain slice,two-electrode voltage clamp and molecular biology methods to elucidate the regulation on N-type Ca²⁺ channels by Sigma-1 receptors.We hope that these results may provide us some clues for our further study on the relationship of Sigma-1 receptors,N-type Ca²⁺ channels and neurotransmitter release.The results are as following:1?It has been reported that there are mainly three subtypes of Ca²⁺ channels in ChIs,namely N-type Ca²⁺ channel,P/Q-type Ca²⁺ channel and L-type Ca²⁺ channel.And N-type Ca²⁺ channels are the most abundant in ChIs.To confirm this conclusion in our study,we used sc RT-PCR to check the transcription of Ca²⁺ channels in 38 ChIs.In our research,there were 92.1%,78.9%,28.9% neurons of 38 ChIs transcribed N-type,P/Q-type and L-type Ca²⁺ channel RNA transcription,respectively.This result was consistent with the previous reports,and indicated that we could choose this neuron for the study of N-type Ca²⁺ channel.2?Combining the previous result and the function of ChIs in controlling ACh release,we selected ChIs as our objects in this study.We should first verify that if there is Sigma-1 receptor expressing in ChIs.By using sc RT-PCR in 30 ChIs,we found that at RNA level there were Sigma-1 receptors in every ChIs.The immune-fluorescence in sequential brain slice showed us Sigma-1 receptors expressed in ChIs at protein level.We proved the expression of Sigma-1 receptors in ChIs for the first time by using sc RT-PCR and immune-fluorescence.This is the foundation for us to study the effect on N-type Ca²⁺ channels by Sigma-1 receptors.3?We recorded the Ca²⁺ currents in dorsal–striatum ChIs by electrophysiology.Specific N-type Ca²⁺ channel blocker w-conotoxin-GVIA?500 n M?blocked the Ca²⁺ currents to 27.7% ± 16.0% of control,which indicated us 72.3%?n=3,P < 0.05?of the currents recorded was N-type Ca²⁺ channels.To investigate whether Sigma-1 receptors could regulate Ca²⁺ channels in ChIs,we used the selective Sigma-1 receptor agonist SKF-10047 at different concentrations in the recording solution.We obtained the currents inhibited by SKF-10047 was 61.3% ± 7.1% of control?n=11,P < 0.05?,71.6% ± 4.9% of control?n=9,P < 0.05?and 106.9% ± 18.2% of control?n=4,P > 0.05?at 50 ?M,25?M and 12.5?M respectively.To ensure the inhibition effect by Sigma-1 receptor agonist,we used another agonist of the receptor,Pre-084.The results showed 50 ?M Pre-084 could inhibit the current to 72.9% ± 6.4% of control?n=7,P < 0.05?.We got that Sigma-1 receptor agonist could inhibit the Ca²⁺ currents in ChIs.To demonstrate the inhibition on Ca²⁺ channel is indeed through Sigma-1 receptors,we added 100 ?M BD-1063 with 50 ?M SKF-10047 together into the recording solution at the same time,and we obtained that the current was 90.0% ± 5.1% of control?n=5,P > 0.05?.These results above suggested that the inhibition on Ca²⁺ channels including N-type Ca²⁺ channels in ChIs was mediated by Sigma-1 receptors.4?Considering the advantages of Xenopus laevis oocytes in researching ion channels,we selected oocytes to study the effect on N-type Ca²⁺ channels by Sigma-1 receptors.First,we injected the c RNA of Sigma-1 receptors or N-type Ca²⁺ channels separately into oocytes as control groups.By two-electrode voltage clamp?TEVC?patch,we obtained the normal N-type Ca²⁺ currents expressed in oocytes.From Western Blot we learned that Sigma-1 receptors had already expressed in oocytes.These results indicated we have established the oocytes expressing system of Sigma-1 receptors and N-type Ca²⁺ channels successfully.5?We should establish the co-expressed system of these two proteins in oocytes to investigate the interaction between them.We injected the mixture of the c RNA at a volume ratio of 1:1 into oocytes,48 h later,we began to record N-type Ca²⁺ currents.Under this condition,we could not obtain the N-type currents.By decreasing the volume of Sigma-1 receptors and remaining the volume of N-type Ca²⁺ channels,the currents we recorded at the ratio of 0.5:1,0.25:1,0.125:1,0.0625:1 and 0.03125:1 was respectively 29.9% ± 3.9%?n = 18,P < 0.001?,52.6% ± 3.8%?n = 17,P < 0.001?,72.1% ± 3.7%?n = 26,P < 0.001?,85.0% ± 5.8%?n = 17,P < 0.05?,89.9% ± 7.5%?n = 10,P > 0.05?of the control currents without Sigma-1 receptors.We concluded from these results that N-type Ca²⁺ currents increased with the decrease of Sigma-1 receptors in oocytes,and this inhibition was ligand-independent.6?To observe whether the Sigma-1 receptors could regulate the N-type Ca²⁺ channels in ligand-dependent manner,we choose the oocytes with the Sigma-1 receptor: N-type Ca²⁺ channel at the ratio of 0.25:1 as our objects.At this ratio,there was not only the expression of Sigma-1 receptors,and we could also record the N-type Ca²⁺ currents at the same time.We used the electrophysiology to study the inhibition by different concentrations of Sigma-1 receptor agonist SKF-10047.The results showed that 1 ?M,5 ?M,25 ?M,50 ?M,100 ?M SKF-10047 could inhibit the N-type Ca²⁺ currents to 94.9% ±1.6%?n=8,P < 0.05?,89.9% ± 1.1% ?n=20,P < 0.005?,83.0% ± 0.7%?n=10,P < 0.001?,73.5% ± 6.5%?n=7,P < 0.001?,67.7% ± 3.7%?n=5,P < 0.001?of control,respectively.To prove the inhibition was mediated by Sigma-1 receptors,50 ?M BD-1063 was added into the recording solution,and it had no effect on the N-type Ca²⁺ channels?the current was 93.2% ± 2.1% of control,n=6,P > 0.05?.When both 50 ?M SKF-10047 and 50 ?M BD-1063 were perfused to the oocytes at the same time,we found BD-1063 could abolish the blockage on N-type Ca²⁺ channels by SKF-10047.These results indicated that the inhibition on N-type Ca²⁺ channels was mediated by Sigma-1 receptors in the same way as in ChIs.7?To test the impact of calcium release channels,we added the specific blockers of these channels into inner solution in recording pipette.The blockers we used were Ca²⁺ chelating agent?20 m M BAPTA?,Ryanodine receptor blocker?10 ?M ruthenium red?and IP3 receptor blocker?100 ?M 2-APB?.With the existence of the blockers,100 ?M SKF-10047 could inhibit the current respectively to 83.4% ± 2.3%?n=6,P < 0.05?,86.9% ± 1.1%?n=7,P < 0.05?,93.9% ± 1.1%?n=5,P < 0.05?of the control,while the SKF-10047 inhibited the current to 67.7% ± 3.7%?n=5,P < 0.05?.8?From these results mentioned above,we hypothesized that there was direct protein-protein interaction between Sigma-1 receptors and N-type Ca²⁺ channels or at least the two proteins located at a very close proximity.To observe the expression and distribution of the two proteins more visualized,we transfected the constructs with fluorescence tags of the Sigma-1 receptors and N-type Ca²⁺ channels transiently into HEK-293 T cells.The red fluorescence indicated the expression of Sigma-1 receptors and green represented the expression of N-type Ca²⁺ channels.With confocal microscopy,in the co-transfected cells we observed the receptors and the channels were expressed successfully.And the images suggested the two proteins may be co-localized in HEK-293 T cells.9? We performed FRET to investigate the co-localization of the Sigma-1 receptors and N-type Ca²⁺ channels.The signal with the area >0.02 mm2,the intensity ?0.5 can be regarded as the position of protein-protein interaction.The results showed that there were 203 positive signals in 11 fields of co-transfected cells.In negative control,no signal met this standard.Our data thus support a close proximity interaction between the Sigma-1 receptors and the N-type Ca²⁺ channels.10? Co-immunoprecipitaion was carried out to verify the existence of the receptor-channel complex further.The stripe indicated that the anti-Sigma-1 receptor antibody or anti-N-type Ca²⁺ channel antibody could pull down the Sigma-1 receptors and the N-type Ca²⁺ channels together.In negative control,no corresponding or specific blot was present.The results demonstrated the direct interaction between the receptors and the channels in HEK-293 T cells.From the results stated above,we learned that the Sigma-1 receptors could express in ChIs for the first time.With the electrophysiological technology,we found Sigma-1 receptors inhibited the N-type Ca²⁺ channels not only in ChIs on the rat brain slice but also in xenopus oocytes in a ligand-dependent manner.In the exogenous system-xenopus oocytes,Sigma-1 receptors could also inhibit the Ca²⁺ channels in a ligand-independent manner,and there was a positive correlation between the inhibition intensity and the expression level of Sigma-1 receptor protein.The molecular biological experiments showed that Sigma-1 receptors and N-type Ca²⁺ channels could form a protein complex in HEK-293 T cells.Based on these results,the negative regulation on N-type Ca²⁺ channels may be due to the protein-protein interaction with Sigma-1 receptors and the receptor's consecutive activity by the agonists.In this way,we presume that Sigma-1 receptor could control the neurotransmitter release by regulating the N-type Ca²⁺ channels' activation or inactivation.These results fill the blank of the influence on N-type Ca²⁺ channels by Sigma-1 receptors,provide us some clues for further study on the relationship of the Sigma-1 receptors,the N-type Ca²⁺ channels and neurotransmitter release in the native tissue or at the system level.And they also offer a new view point for treating the diseases relevant with N-type Ca²⁺ channels.