Study On The Relationship Between PIP5K Isoform And PIP₂ And M Current Regulation In Rat SCG Neurons

M current was first discovered in bullfrog sympathetic neurons by Brown and Admas in 1980. It is a slowly activating, non-inactivating, voltage-dependent potassium current. It is named M current because of its suppression by muscarinic receptor activation. M current is wildly distributed in neuronal system, and is one of the major mechanisms controlling the neuronal excitability. M current is the only current activated around threshold potential of neurons and its functional depression could lead to the increased excitability of the neurons, leading to the diseases such as epilepsy. So far many neuronal transmitters and peptides have been found to modulate the M currents.The G-protein coupled receptors (GPCRs) account for the majority of membrane receptors, and have important pathophysiological and pharmacological significance. Different GPCRs respond to a wide variety of different external stimulants and initiate a wide spectrum of intracellular responses by activating a number of different G proteins. All G proteins are composed of threeα,β,γsubunits. G proteins can be divided into four sub-families: G_s,G_i/o,G_q and G₁₂, according to theαsubunit contained.Ample evidence demonstrate that activation of G_q protein-coupled-receptors will result in M current inhibition. After activation of G_q protein-coupled-receptor, activated PLC hydrolyze PIP₂, yielding two intracellular second messengers IP₃ and DAG. IP₃ then trigger calcium release from endoplasmic reticulum. DAG activates PKC. Through years of research, it is gradually realized that the membrane PIP₂ hydrolysis, intracellular calcium increase may play an important role in receptor-mediated M current suppression.PIP₂ is an important phospholipid that modulates multiple cellular processes. In mammalian cells, phosphatidylinositol 4-phosphate 5-kinase (PIP5K) generates phosphatidylinositol 4,5-bisphosphate (PI4,5P₂, PIP₂) by catalyzing the phosphorylation of PI4P at the D5 position of the inositol ring. To date, three mammalian PIP5K isozymesα,βandγ, and three splicing variants of theγisozyme,γ635,γ661, andγ687, have been identified.It is not clear whether there is a specificity of PIP5K isozymes in controlling the PIP₂ metabolism. For this purpose, we used techniques of primary neuron transfection, small RNA interference, laser scanning confocal microscopy, western blot, immunofluorescence, intracellular calcium imaging and patch clamp, to study the role of PIP5K isozymes in PIP₂ metabolism and M potassium current modulation in rat SCG neurons. The study is divided into five parts: (1) Electrotransfection of rat SCG neurons; (2) Expression of PIP5K isoforms in rat SCG neurons and interference efficiency of PIP5K isoform specific RNAi; (3) PIP5K isoform related PIP₂ distribution and hydrolysis in rat SCG neurons; (4) PIP5K isoform related calcium signaling in rat SCG neurons; (5) PIP5K isoform related M current regulation in rat SCG neurons.Part 1 Electrotransfection of rat SCG neuronsObjective: To establish a high efficient method for electrotransfection of large numbers of SCG neurons from rats with different ages.Methods: SCG neurons from 7, 14 and 40 days old SD rat were cultured in unmodified or modified condition and observed 24 hours after Nucleofector transfection. The cell survival rate determined on the basis of trypan blue dye exclusion was studied. The transfection conditions were optimized by changing the ratio of plasmid DNA/siRNA and nucleofector solution. The transfection efficiency or interference efficiency in 24 hours after transfection were assessed by using confocal microscopy.Results: (1) SCG neurons from SD rats older than 14 days had a survival rate of greater than 75% in modified culture medium after 24 hours of transfection, which was markedly higher compared to unmodified culture medium (P < 0.01). The results were consistent and the transfected cells were in good condition necessary for the follow-up experiments. (2) Higher transfection efficiency or interference efficiency was obtained after using optimized transfect conditions:ratio of DNA and nucleofector solution is 1:100 (μg :μl) for DNA transfection; ratio of siRNA and nucleofector solution is 2:100 (μg :μl) for siRNA interference.Conclusions: (1) By using the Nucleofector^TM transfection system with G-013 and Rat Neuron Nucleofector Kit (lot: VPG-1003), SCG neurons from rats of different ages can be successfully transfected with DNA or siRNA. (2) The L-15 culture medium with 20% FBS and 25 ng/ml NGF 7S improved survival rate of transfected SCG neurons from rats older than 14 days significantly. (3) The optimized transfection conditions for DNA/siRNA and nucleofector solution ratio were found and could be used for further study.Part 2 Expression of PIP5K isoforms in rat SCG neurons and interference efficiency of PIP5K isoform specific RNAiObjective: To investigate the expression and distribution of PIP5K and study the interference efficiency of PIP5K isoform specific RNAi.Methods: RT-PCR, western blot and immunofluorescence were used to detect the expression and distribution of PIP5K isoformα,βandγ; isoform specific siRNA was used to depress PIP5K; western blot method was used to detect the interference efficiency of siRNA.Results: (1) Western blot and immunofluorescence results showed that the expression of PIP5Kα,βandγisoforms could not be detected in SCG from 7 days old rats. Western blot, RT-PCR and immunofluorescence results showed PIP5Kα,γisoforms could be detected in SCG from 14 days old rats with distinct cellular locations: PIP5Kαwas mainly distributed on the cell membrane, whileγisoform was found in the membrane and in the cytosol; PIP5Kβisoform could not be detected in SCG from 14 days and 40 days old rats, but was found in hippocampus from 14 and 40 days old rat. (2) Both PIP5KαRNAi and PIP5KγRNAi showed the interference efficiency more than 50% when assessed by western blot and immunofluorescence methods.Conclusions: (1) PIP5Kαandγisoforms are expressed in SCG from 14 day-old rat. PIP5Kαisoform is mainly distributed in the cell membrane, and theγisoforms is more widely distributed. The expression of PIP5Kβis not found in SCG of rat. (2) siRNA for PIP5Kαandγisoforms have relatively high efficiency in depressing the expression of PIP5Kαandγisoforms. Part 3 PIP5K isoform related PIP₂ distribution and hydrolysis in rat SCG neuronsObjective: To study the relationship between the PIP5K isoform and distribution and content of PIP₂ in rat SCG neurons. To study the relationship between the PIP5K isoform and PIP₂ hydrolysis induced by activation of M receptor.Methods: PLC-PH-GFP was used to observe the location of PIP₂ in SCG neurons. Anti-PIP₂ immuonfluorescence method was used to assess the PIP₂ content. siRNA for PIP5K isoform was used to depress the expression of PIP5K isoforms.Results: (1) PIP5KαRNAi greatly affected the distribution and content of PIP₂ in rat SCG neurons. PIP5KγRNAi did not affect the cell PIP₂ distribution but lowered membrane PIP₂ content. (2) Negative control siRNA group caused a translocation of the fluorescent probe from the membrane to cytosol. PIP5Kαand PIP5KγRNAi abolished the translocation of PLC-PH-GFP induced by activation of M receptor.Conclusions: (1) PIP5Kαis important for the localized distribution of PIP₂ and both PIP5Kαand PIP5Kγare important for the maintaining of PIP₂ content in the membrane of rat SCG neurons. (2) PIP5Kαand PIP5Kγrelated PIP₂ contribute significantly to the pool of PIP₂ sensitive to M receptor-induced hydrolysis.Part 4 PIP5K isoform related calcium signaling in rat SCG neuronsObjective: To investigate the effect of PIP5Kαandγisoform RNAi on BK-induced calcium signaling in rat SCG neurons.Methods: Neuron PIP5K isoform-specific siRNA was used to depress PIP5K expression. The intracellular Ca²⁺ changes were monitored using dye Fluo-4/AM and confocal microscopy. Results: (1) In the cells transfected with the negative control siRNA, over 68% (34/50) cells showed obvious calcium increase responding to BK; in the cells transfected with the PIP5Kαor PIP5KγsiRNA, about 42% (24/55) and 10% of the neurons (5/51) responded to BK. (2) The magnitudes of the Ca²⁺ increase were also compared among three groups. The ratio of the calcium concentrations before and after BK application in negative control siRNA group, PIP5KαsiRNA group and PIP5KγsiRNA group were 1.76, 1.45 and 1.09, respectively. The ratio of PIP5KγsiRNA group was significantly less than that of negative control siRNA (P < 0.01) and PIP5KαsiRNA group (P < 0.05).Conclusions: PIP5KγsiRNA significantly affects elevation of intracellular Ca²⁺ induced by BK in SCG cells, suggesting that PIP5Kγisoform generates the PIP₂ that is involved in the receptor-induced Ca²⁺ transients.Part 5 PIP5K isoform related M current regulation in rat SCG neuronsObjective: To study the effect of PIP5K isoform specific RNAi on receptor-mediated M current suppression in rat SCG neurons.Methods: Neuron PIP5K isoform specific siRNA was used to depress PIP5K expression. The M currents in SCG neurons were recorded using the patch clamp technique.Results: (1) BK and Oxo-M, through activation of BK receptor and M receptor, respectively, inhibited the M current recorded from SCG neurons. For BK, it inhibited the M currents recorded from the negative control siRNA SCG cells,the PIP5KαsiRNA SCG cells and PIP5KγsiRNA SCG cells by 79.94±7.67%,97.74±1.52% and 57.26±8.03%, respectively. The inhibition in PIP5KγsiRNA SCG cells was significantly less than that in negative control siRNA and PIP5KαsiRNA SCG cells (P < 0.05). (2) For Oxo-M, it inhibited the M currents from the negative control siRNA SCG cells,the PIP5KαsiRNA SCG cells and PIP5KγsiRNA SCG cells by 93.27±3.61%,95.81±4.00% and 48.04±4.24%, respectively. The inhibition in PIP5KγsiRNA SCG cells was significantly less than that in negative control siRNA and PIP5KαsiRNA SCG cells (P < 0.05).Conclusions: PIP5KγRNAi significantly affects BK and Oxo-M induced M currents inhibition in SCG cells, suggesting that PIP5Kγisoform generates the PIP₂ that is involved in the maintaining of M channel function in SCG neurons.SUMMARYIn this study, we studied the expression of different PIP5K isoforms in SCG neurons and their role in PIP₂ dynamics and related functions—intracellular Ca2+ transients and M current modulation in rat SCG neurons. On the basis of a well optimized method for transfecting SCG neurons with interference RNA for different PIP5K isoforms, the roles of these PIP5K isoforms were convincingly assessed. In rat SCG neurons, we only identified PIP5Kαand PIP5Kγisoforms in SCG neurons; it seems that PIP5Kβis not present in rat SCG neurons. It seems clear from this study that the PIP₂ generated by PIP5Kγplays a more dominated role in receptor-induced intracellular Ca2+ transients and M current inhibition.The specificity of PIP5K isoforms in generating PIP₂ suggests that the cellular PIP₂ metabolism is specifically regulated. Thus both the synthesis as well as the hydrolysis of PIP₂ is regulated through delicate specific mechanisms. This specificity has important physiological significance and may serve as one key component in specificity of cell signaling.