Effects Of PICK1 Knockout On The Functions Of ASICs And Related Mechanisms

PartⅠEffects of PICK1 gene knockout on the functions and expressions of ASICsAim:Acid-sensing ion channels (ASICs), with the character of being activated by a drop of the extracellular pH or an increase of proton concentration, are one of the members of the degenerin/epithelial Na+ channel superfamily (DEG/ENaC). Four genes (ASIC1-ASIC4) encoding six subunits have been identified to date, they are ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4. Among them, ASIC1a and ASIC2a seem to be the most important subunits in the brain, including physiological conditions and pathological processes. PICK1 (protein that interacts with C kinase 1) is recently shown to be one of the partner proteins interacting with ASICs, especially ASIC1 and ASIC2. ASIC1 and ASIC2 have been displayed to co-localize with PICK1 at the peripheral sensory endings of dorsal root ganglia neurons, as well as the synapse and cell bodies of some central neurons. PICK1 interacts specifically with the C-termini of these ASICs through its PDZ domain. But the direct evidences to investigate the mutual relationships between PICK1 and ASICs have not been found.Methods: The effects of PICK1 gene on the functions and expressions of ASICs in cultured cortical neurons were observed by using PICK1 knockout mice, together with the whole-cell patch clamp and calcium imaging techniques, RT-PCR,western blotting and immunofluorescent methods.Results: The functions of ASICs decreased significantly, while the expressions remained unchanged after PICK1 knocking out.1. Effects of PICK1 gene knockout on the functions of ASICsA. The characteristics of ASIC currents in cultured mice cortical neurons. Using a whole-cell recording at a holding potential of -80 mV, a transient inward current was evoked by a rapid lowering of pH from 7.4 to 6.0. This current had a nearly linear I-V relationship, and could be almost blocked in the presents of amiloride 100μM.B. ASIC currents of littermate mice cortical neurons decreased after PICK1 knocked out. The amplitudes were 224.2±17.59 pA (n=15) in widetype, 140.3±28.55 pA (n=17, p<0.05 vs WT) in heterozygote, and 61.3±9.62 pA (n=14, p<0.01 vs WT, p<0.05 vs HT) in homozygote, respectively.C. The characteristics of [Ca2+]i elevation via ASICs in primary cultured cortical neurons. An elevation of [Ca2+]i were observed when extracellular acidic solution (pH=6.0) was applied to the system.Remained [Ca2+]i was elevated during prolonged perfusion of low pH solutions, and fell-off after peaking. Amiloride 100μM partly blocked the elevation, and 500μM almost abolished it.D. The acid-induced increasing of [Ca2+]i in littermate cortical neurons were down-regulated by PICK1 knocked out. The△[Ca2+]/[Ca2+] were 0.708±0.070 (n=20) in widetype, 0.333±0.023 (n=27, p<0.01 vs WT) in heterozygote, and 0.195±0.020 (n=26, p<0.01 vs WT, p<0.01 vs heterozygote) in homozygote, respectively.2. Effects of PICK1 gene knockout on the expressions of ASICsA. The mRNA and proteins of ASIC1 and ASIC2a were unchanged after PICK1 knockout. Consistent with PICK1+/+ mice, ASIC1, 2a transcripts were also detected in PICK1+/- and -/- mice. By comparing to the control ofβ-actin, the genes of ASIC1 and ASIC2a were almost not changed among these three types of animals (n=3). The protein levels of ASIC1, as well as ASIC2a, maintained unchanged in PICK1-KO mice (n=6) by western blotting.B. There is a trafficking of ASIC1 and ASIC2a after PICK1 depletion. Although the total fluorescent density were almost the same in WT and KO group by the immunocytochemical analysis, the distribution of ASIC1 and ASIC2a in the membrane seemed to be reduced in KO group.Conclusion:1. The ASICs currents of cultured cortical neurons, as well as the level of intracellular Ca2+ concentrations induced by extracellular acidosis, were weakened after PICK1 gene disrupted. Since the ASICs currents were elicited by pH dropping to 6.0 in our experiment, and it's ASIC1a permeable to Ca2+, we speculated that the modulation effects of PICK1 on ASICs currents and acid-inducing Ca2+ increasing were mainly through homomeric ASIC1a channels, ASIC2a associating with ASIC1a as the heteromultimeric channels might also be involved in it, but homomeric ASIC2a channels seemed to be influenced little.2. The total mRNA and protein levels of ASIC1 and ASIC2a in cortical neurons didn't alter, but the protein distributions of ASIC1 in cell membrane were reduced in PICK1 null cortices. So, the mechanism that PICK1 affacted ASIC functions may lie in the trafficking of ASICs with disruption of PICK1.PartⅡEffects of PICK1 gene knockout on the modulation of ASIC functions with protein kinase CAim: Although many modulators were clarified to regulate ASICs up to date, including extracellular and endogenous elements, protein kinase C were one of the common upstream elements to modulate ASICs. ASICs could be phosphorylated by PKC acting on their phosphorylation sites, which related to the role of PICK1. If PICK1 had a certain effect on the functions of ASICs, what's the possible mechanism? We need to further explore the interactions among PKC, PICK1 and ASICs here. Methods: To observe the effects of activator and inhibtor of PKC on ASIC functions by using the whole cell patch clamp, calcium imaging, western blotting and immunofluorescent techniques.Results:1. PKC up-regulated ASIC currents in cultured mice cortical neurons via PICK1. The ASIC currents were induced with the amplitude of 228.60±20.09pA (n=13) in WT and 65.40±6.96pA (n=10) in KO neurons, respectively. Preincubation of 10μM GF109203X (PKC inhibitor) and 1μM PMA (PKC activator) for 30 min markedly altered the amplitude of the currents in WT group excluding KO group. GF109203X decreased the current approximately 1- fold to 107.50±15.75 pA (n=19, p<0.01 vs control), and PKC activator, PMA, increased it more than 3- folds to 756.9±144.9 pA (n=11, p<0.01 vs control) in WT cortical neurons. Meanwhile, the variations of ASICs currents in KO mice were not significant, remaining at 64.4±4.81 pA (n=7, p>0.05 vs control) and 84.3±13.05 pA (n=10, p>0.05 vs control), respectively.2. PKC promoted elevation of [Ca2+]i induced by acids in cultured mice cortical neurons via PICK1. An elevation of [Ca2+]i was seen when extracellular acidic solution (pH=6.0) was applied, the normalized variation of [Ca2+]i (△F/F) was 0.662±0.062 (n=17) in WT group and 0.228±0.016 (n=33) in KO group. Pretreatment with 10μM GF109203X obviously blocked the increasing of [Ca2+]i and 1μM PMA augmented it in WT neurons, while the changes of normalized△[Ca2+]i were not significant among three groups in KO neurons. The△F/F reduced to 0.175±0.014 (n=26, p<0.01 vs control) with PKC inhibitor and raised to 1.08±0.112 (n=14, p<0.01 vs control) with PKC activator in WT group, however, it remained at 0.218±0.024 (n=20, p>0.05 vs control) and 0.267±0.025 (n=13, p>0.05 vs control) respectively in KO group.3. Knockout PICK1 gene neither changed PKC expression nor PKCαprotein level. Both the western blotting and staining images showed no significant change of PKC/PKCαexpressions, the distributions of PKCαbetween WT and PICK1-KO mice cortical neurons were also the same, both membrane and cytoplasm of neuron expressed extensive PKCα.Conclusions:1. The up-regulation of PKC on ASIC currents and elevations of [Ca2+]i induced by extracelluar acid solution depended on the role of PICK1 gene, PICK1 was the"bridg"between PKC and ASICs.2. The specific actions on wildtype cortical neurons with agents to activate or inhibit PKC were independent on the quantity of PKC expression, which further identified the linkage of PICK1 between PKC and ASICs.PartⅢEffects of PICK1 gene knockout on the modulation of ASIC functions with protein kinase AAim: Protein kinase A was another common upstream element to modulate ASICs. ASICs could also be phosphorylated by PKA acting on their phosphorylation sites, which related to the role of PICK1. We also need to further explore the interactions among PKA, PICK1 and ASICs here.Methods: To observe the effects of activator and inhibtor of PKA on ASIC functions by using the whole cell patch clamp, calcium imaging, western blotting and immunofluorescent techniques.Results:1. Down-regulation of PKA on ASIC functions in WT neurons. The ASIC currents were induced with the amplitude of 224.2±17.59 pA (n = 15) in WT neurons, Preincubation of 10μM forskolin(PKA activator)reduced the amplitude to 108±14.67 pA (n=9, P<0.001 vs control),5μM PKA inhibitor fragment 6-22 amide(PKA inhibitor) raised it to 306.3±22.9 pA (n=12, P<0.01 vs control)。The normalized variation of [Ca2+]i (△F/F) in WT neurons was similar to that of currents, the△F/F in control group was 0.708±0.07 (n=20),0.140±0.013 (n=17,P<0.001 vs control) in 10μM forskolin treated group, and 0.978±0.108 (n=19,P<0.05 vs control) in 5μM PKAI group。2. The regulations of PKA on ASIC functions were weakened in KO neurons. The ASIC currents were induced with the amplitude of 61.2±9.62 pA (n=14) in KO neurons, Preincubation of 10μM forskolin decreased the amplitude to 36.15±10.15pA (n=10, P<0.05 vs control), but 5μM PKA inhibitor fragment 6-22 amide had almost no effect on the amplitudes, remained with 60.82±11.43 pA (n=11, p>0.05 vs control)。The varivation of△F/F in KO neurons was similar to that of currents, the value in control group was 0.195±0.02 (n=26),0.157±0.011 (n=20, p <0.05 vs control) in 10μM forskolin treated group, and 0.170±0.0218 (n=28,p>0.05 vs control) in 5μM PKAI group.3. Knockout PICK1 gene did not change the total PKA protein level.4. PKA didn't affect the expressions of ASIC1. Preincubation with forskolin and PKAI for 24h,the total fluorescence density were the same as control (p>0.05 vs control).Conclusions:1. PKA down regulated the functions of ASIC in WT primary cortical neurons (ASIC currents and elevations of [Ca2+]i inducing by extracelluar acid solution), the effects were weakened in KO mice. So, the modulations depended on the induction of PICK1 partly, which was not the only role. There may be a direct effect or others between PKA and ASICs.2. The expressions of PKA remained unchanged after PICK1 knockout, so it's not the number of PKA that affected the functions of ASIC1 and ASIC2a.3. Activator and inhibitor of PKA had no contributions to the expressions of ASICs in WT cortical neurons, so the direct role of PKA may employ at other facts, not affecting the number of ASICs.Summary:1. The ASICs currents of cultured cortical neurons, as well as the level of intracellular Ca2+ concentrations induced by extracellular acidosis, were weakened after PICK1 gene disrupted, the mechanisms may lie in the trafficking of ASICs with disruption of PICK1.2. The up-regulation of PKC on ASIC currents and elevations of [Ca2+]i induced by extracelluar acid solution depended on the role of PICK1 gene, PICK1 was the"bridg" between PKC and ASICs. The disappearance of up-regulation was related to the depletion of PICK1, not the alteration of PKC expressions.3. The down-regulation of PKA on ASIC currents and elevations of [Ca2+]i induced by extracelluar acid solution depended on the role of PICK1 gene partly, the direct effects on ASICs by PKA or other pathway may also participart in it. The blocking of down-regulation of PKA in KO mice did not lie on the total expressions of PKA, but on the disappering of PICK1 induction.