PICK1基因敲除对ASICs功能的影响及其机制
|
摘要
第一部分PICK1基因敲除对ASICs通道功能和表达的影响
目的:酸敏感离子通道(acid-sensing ion channels, ASICs)是ENaC/DEG (epithelial Na+ channel/degenerin)通道超家族的成员之一,能被细胞外pH下降或H+浓度上升激活。目前已克隆出的ASICs主要有ASIC1a、ASIC1b、ASIC2a、ASIC2b、ASIC3和ASIC4。其中,ASIC1a和ASIC2a是大脑内最重要的两个亚单位,在生理和病理过程中发挥重要作用。PICK1 (protein that interacts with C kinase 1)是ASICs的锚定蛋白,ASIC1和ASIC2与PICK1在背根神经节神经元的外周感觉神经末梢上有共表达,同样也在某些中枢神经元的突触和胞体内有共表达,PICK1还通过其PDZ区域特异性的与ASICs蛋白的C-末端作用。但到目前为止,还没有直接证据证明PICK1对ASICs表达和功能的影响。
方法:应用PICK1基因敲除小鼠,采用全细胞膜片钳和钙影像技术,RT-PCR、western blotting和细胞免疫荧光方法,观察PICK1基因敲除对原代培养的皮质神经元ASICs功能的影响,以及皮质上ASICs基因和蛋白表达的影响。
结果:PICK1基因敲除后,ASICs功能发生明显改变,而通道的表达不变。
1. PICK1基因敲除对ASICs通道功能的影响。
⑴原代培养的皮质神经元ASICs电流的特点。形成全细胞模式后,在电压钳模式下,保持电位-80 mV,采用多管灌流系统,将细胞旁外液的pH值由7.4迅速转换成6.0,可诱发出一短暂、快速激活的内向电流,该电流随电压的减少,幅值不断减少;100μM的阿米洛利几乎完全阻断此内向电流。
⑵PICK1基因敲除后ASICs电流幅值减小。培养同窝的PICK1基因敲除C57/BL6小鼠皮质神经元并记录ASICs电流,野生型ASICs电流的幅值为224.2±17.59 pA (n=15),杂合子为140.3±28.55 pA (n=17, p<0.05 vs WT),纯合子为61.3±9.62 pA (n=14, p<0.01 vs WT, p<0.05 vs HT)。
⑶原代培养的皮质神经元ASICs通道对钙具有通透性。当细胞外液由标准脑脊液迅速转换成pH 6.0的酸性外液后可见细胞内[Ca2+]水平的上升,并随酸性溶液的灌注继续增加,直至达峰值,而后逐渐下降,恢复基线。100μM的阿米洛利可部分阻断酸所诱发的细胞内[Ca2+]水平的上升,500μM则可完全阻断,从而证实该效应为ASICs诱导。
⑷PICK1基因敲除后酸诱导的胞内钙增加减少。取同窝培养的PICK1基因敲除C57/BL6小鼠皮层神经元,记录ASICs诱导的胞内钙增加情况,野生型△[Ca2+] / [Ca2+]比值为0.708±0.070 (n=20),杂合子为0.333±0.023 (n=27, p<0.01 vs WT),纯合子为0.195±0.020 (n=26, p<0.01 vs WT, p<0.01 vs HT)。
2. PICK1基因敲除对ASICs通道表达的影响。
⑴PICK1基因敲除后,ASIC1和ASIC2a的mRNA和蛋白表达均不变。野生型C57/BL6小鼠皮质表达丰富的ASIC1和ASIC2a基因,PICK1基因敲除小鼠的皮质也表达ASIC1和ASIC2a基因,与野生型相比,其含量无明显变化(n=3);同样,野生型与纯合子相比,ASIC1和ASIC2a的蛋白表达也无明显差异(n=6)。
⑵PICK1基因敲除后,ASIC1和ASIC2a在细胞膜上的分布减少。免疫荧光的统计结果显示,尽管总的荧光密度无明显改变,但PICK1基因敲除小鼠的皮质神经元,细胞膜上ASIC1和ASIC2a的表达没有野生型完整和连续,荧光强度减弱。
结论:
1. PICK1基因敲除后,培养的小鼠皮质神经元ASIC电流幅度减少,酸诱导的胞内钙增加也减少。因为在pH 6.0的实验条件下,主要是激活ASIC1a同聚体电流,而且在中枢神经元中,只有ASIC1a同聚体被激活后才对Ca2+通透,推测PICK1基因增加ASICs的功能主要是通过ASIC1a来实现的,ASIC2a也部分参与其中。
2. PICK1影响ASICs功能的机制并不在于PICK1敲除后ASIC1或ASIC2a的基因和蛋白表达水平减少,而可能是PICK1的缺失导致ASICs发生定位的改变,其蛋白从膜上转运到胞浆内所致,即ASICs的下膜作用。
第二部分PICK1基因敲除对蛋白激酶C调节ASICs功能的影响
目的:ASICs的调控受很多因素的影响,包括细胞外和细胞内因素。蛋白激酶PKC是常见的调节ASICs功能的上游因子之一,ASICs上有PKC的磷酸化位点。而PICK1又以其特定的PDZ区域与ASIC1a、ASIC2a和ASIC2b的C末端结合,故上述作用很多都与PICK1有关。既然在上文中我们已经证实,PICK1对ASICs功能的发挥起到重要作用,其基因的缺失会影响ASICs电流以及钙的通透性,那么,其进一步的机制是否与蛋白激酶C有关?PKC、PICK1和ASICs之间是一个怎样的关系和相互作用?这些都值得我们去进一步探讨。
方法:采用全细胞膜片钳、钙影像技术,western blotting和细胞免疫荧光技术,观察PICK1基因敲除前后,PKC激动剂和抑制剂对ASICs功能如何影响,探讨可能的机制。
结果:
1. PKC对小鼠皮质神经元ASICs电流有正性调节作用,该作用依赖于PICK1基因。在给予PKC激动剂或抑制剂前,ASICs电流的幅值在野生型小鼠神经元为228.60±20.09pA (n=13),纯合子为65.40±6.96pA (n=10)。当分别给予10μM GF109203X(PKC抑制剂)和1μM PMA(PKC激动剂)预孵育30 min后,野生型小鼠的电流幅值分别下降为107.50±15.75 pA (n=19, p<0.01 vs control),和增加为756.9±144.9 pA (n=11, p<0.01 vs control)。而在纯合子小鼠组,与对照组相比,无论是PKC抑制剂还是PKC激动剂对ASICs的影响均不明显,其电流值分别维持在64.4±4.81 pA (n=7, p>0.05 vs control )和84.3±13.05 pA (n=10, p>0.05 vs control)。
2. PKC对小鼠皮质神经元ASICs介导的钙升高有正性调节作用,该作用也依赖于PICK1的介导。细胞外酸性环境(pH=6.0)诱导出的瞬时钙升高,野生型对照组△F/F为0.662±0.062 (n=17),而纯合子为0.228±0.016 (n=33)。分别给予10μM GF109203X和1μM PMA预孵育后,野生型小鼠钙升高发生了明显改变,即GF109203X使△F/F下降至0.175±0.014 (n=26, p<0.01 vs control),PMA则使△F/F升高到1.08±0.112 (n=14, p<0.01 vs control)。而纯合子小鼠组与对照组相比,无论是PKC抑制剂还是PKC激动剂对△F/F的影响均不明显,其值分别维持在0.218±0.024 (n=20, p>0.05 vs control)和0.267±0.025 (n=13, p>0.05 vs control)。
3. PICK1基因敲除后,PKC的蛋白表达和分布不变。Western blotting和免疫荧光的结果均显示,小鼠皮质上无论是总PKC还是特异性与PICK1作用的PKCα的蛋白表达量都未发生改变;PKCα在细胞膜上和胞浆内均匀分布。
结论:
1. PKC对皮质神经元ASICs电流和ASICs介导的胞内钙增加的正性调节作用依赖于PICK1基因的存在,PICK1在其中起到了桥梁作用。
2. PICK1基因敲除后小鼠皮质上PKC和PKCα的蛋白表达总量和分布不变。进一步说明,PKC是通过PICK1的作用调节ASICs通道,PICK1敲除后,这一连接消失,而导致其调节ASICs的功能减弱。
第三部分PICK1基因敲除对蛋白激酶A调节ASICs功能的影响
目的:蛋白激酶PKA是除PKC外的另一常见的调节ASICs功能的上游因子,ASICs上也有PKA的磷酸化位点,能被其磷酸化,并与PICK1有关。PICK1基因的缺失会影响ASICs电流以及钙的释放,那么,其进一步的机制是否与蛋白激酶A也有关?PKA、PICK1和ASICs之间是一个怎样的关系和相互作用?这些也值得我们去进一步探讨。
方法:采用全细胞膜片钳、钙影像技术,western blotting和细胞免疫荧光技术,观察PICK1基因敲除前后,PKA激动剂和抑制剂对ASICs功能如何影响,探讨可能的机制。
结果:
1. PKA对小鼠皮质神经元ASICs电流及ASICs介导的胞内钙增加有负性调节作用。在给予PKA激动剂或抑制剂前,野生型ASICs电流幅值约为224.2±17.59 pA (n = 15),10μM forskolin(PKA激动剂)与神经元共同孵育30 min后,使电流幅值减小为108±14.67 pA (n=9, P<0.001 vs control),5μM PKA inhibitor fragment 6-22 amide(PKA抑制剂,PKAI)则增加ASICs电流,其值为306.3±22.9 pA (n=12, P<0.01 vs control)。酸诱导的钙升高结果,对照组△F/F值为0.708±0.07 (n=20),10μM forskolin使其减少为0.140±0.013 (n=17,p<0.001 vs control),而5μM PKAI使其增加为0.978±0.108 (n=19,p<0.05 vs control)。
2. PICK1基因敲除后,PKA对小鼠皮质神经元ASICs功能的负性调节作用减弱。用培养的PICK1-/-小鼠皮质神经元,观察forskolin和PKAI对ASICs电流的影响。对照组的电流幅值为61.2±9.62 pA (n=14),当分别与10μM forskolin和5μM PKAI共同孵育30 min后,仅forskolin使电流值减少约40%,为36.15±10.15pA (n=10, p<0.05 vs control),用PKAI后电流值仍然维持在和60.82±11.43 pA (n=11, p>0.05 vs control)。酸诱导皮质神经元钙内流增加的改变结果也与ASICs电流的变化一致,对照组△F/F值为0.195±0.02 (n=26),forskolin使其值降为0.157±0.011 (n=20,p<0.05 vs control),PKAI与神经元共同孵育30 min后,其值仍维持在0.170±0.0218 (n=28,p>0.05 vs control)。3. PICK1基因敲除后,皮质上PKA蛋白表达总量未发生改变。4. PKA对ASICs表达无明显影响,在加入forskolin和PKAI孵育24小时后,神经元总的荧光密度不变(p>0.05 vs control)。
结论:
1. PKA对培养的皮质神经元ASICs功能(ASIC电流和ASICs诱导的钙升高)产生负性调节作用;而PICK1基因敲除后,这种调节作用减弱。即PKA对ASICs功能的负性调节仍然依赖PICK1的作用,但并非全部,可能存在PKA的直接作用或其它影响因素。
2. PICK1基因敲除后小鼠皮质上PKA的蛋白表达总量不变,故并非PKA的数量影响了ASIC1和ASIC2a的功能。
3. PKA激动剂和抑制剂对野生型小鼠皮质神经元ASICs的表达无影响,故PKA的直接作用并未体现在对ASICs通道数量的改变,可能有其它因素。
总结:
1. PICK1基因敲除后,小鼠皮质神经元ASICs的功能减弱,其机制可能是ASICs定位发生改变,导致其在细胞膜上的分布减少所致。
2. PKC对皮质神经元ASICs的功能有正性调节作用,该作用的发挥依赖于PICK1基因。基因敲除后,正性调节的消失与PICK1的缺失有关,而不是PKC蛋白总量的改变。
3. PKA对皮质神经元ASICs的功能有负性调节作用,该作用的发挥部分依赖于PICK1基因的存在,可能还有自身直接作用或其它影响因素存在。基因敲除后,PKA蛋白表达总量不变,但PICK1作用的消失使PKA的负性调节作用受抑制。
PartⅠEffects of PICK1 gene knockout on the functions and expressions of ASICs
Aim: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 ASICs
A. 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 ASICs
A. 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 C
Aim: 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 A
Aim: 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.
目的:酸敏感离子通道(acid-sensing ion channels, ASICs)是ENaC/DEG (epithelial Na+ channel/degenerin)通道超家族的成员之一,能被细胞外pH下降或H+浓度上升激活。目前已克隆出的ASICs主要有ASIC1a、ASIC1b、ASIC2a、ASIC2b、ASIC3和ASIC4。其中,ASIC1a和ASIC2a是大脑内最重要的两个亚单位,在生理和病理过程中发挥重要作用。PICK1 (protein that interacts with C kinase 1)是ASICs的锚定蛋白,ASIC1和ASIC2与PICK1在背根神经节神经元的外周感觉神经末梢上有共表达,同样也在某些中枢神经元的突触和胞体内有共表达,PICK1还通过其PDZ区域特异性的与ASICs蛋白的C-末端作用。但到目前为止,还没有直接证据证明PICK1对ASICs表达和功能的影响。
方法:应用PICK1基因敲除小鼠,采用全细胞膜片钳和钙影像技术,RT-PCR、western blotting和细胞免疫荧光方法,观察PICK1基因敲除对原代培养的皮质神经元ASICs功能的影响,以及皮质上ASICs基因和蛋白表达的影响。
结果:PICK1基因敲除后,ASICs功能发生明显改变,而通道的表达不变。
1. PICK1基因敲除对ASICs通道功能的影响。
⑴原代培养的皮质神经元ASICs电流的特点。形成全细胞模式后,在电压钳模式下,保持电位-80 mV,采用多管灌流系统,将细胞旁外液的pH值由7.4迅速转换成6.0,可诱发出一短暂、快速激活的内向电流,该电流随电压的减少,幅值不断减少;100μM的阿米洛利几乎完全阻断此内向电流。
⑵PICK1基因敲除后ASICs电流幅值减小。培养同窝的PICK1基因敲除C57/BL6小鼠皮质神经元并记录ASICs电流,野生型ASICs电流的幅值为224.2±17.59 pA (n=15),杂合子为140.3±28.55 pA (n=17, p<0.05 vs WT),纯合子为61.3±9.62 pA (n=14, p<0.01 vs WT, p<0.05 vs HT)。
⑶原代培养的皮质神经元ASICs通道对钙具有通透性。当细胞外液由标准脑脊液迅速转换成pH 6.0的酸性外液后可见细胞内[Ca2+]水平的上升,并随酸性溶液的灌注继续增加,直至达峰值,而后逐渐下降,恢复基线。100μM的阿米洛利可部分阻断酸所诱发的细胞内[Ca2+]水平的上升,500μM则可完全阻断,从而证实该效应为ASICs诱导。
⑷PICK1基因敲除后酸诱导的胞内钙增加减少。取同窝培养的PICK1基因敲除C57/BL6小鼠皮层神经元,记录ASICs诱导的胞内钙增加情况,野生型△[Ca2+] / [Ca2+]比值为0.708±0.070 (n=20),杂合子为0.333±0.023 (n=27, p<0.01 vs WT),纯合子为0.195±0.020 (n=26, p<0.01 vs WT, p<0.01 vs HT)。
2. PICK1基因敲除对ASICs通道表达的影响。
⑴PICK1基因敲除后,ASIC1和ASIC2a的mRNA和蛋白表达均不变。野生型C57/BL6小鼠皮质表达丰富的ASIC1和ASIC2a基因,PICK1基因敲除小鼠的皮质也表达ASIC1和ASIC2a基因,与野生型相比,其含量无明显变化(n=3);同样,野生型与纯合子相比,ASIC1和ASIC2a的蛋白表达也无明显差异(n=6)。
⑵PICK1基因敲除后,ASIC1和ASIC2a在细胞膜上的分布减少。免疫荧光的统计结果显示,尽管总的荧光密度无明显改变,但PICK1基因敲除小鼠的皮质神经元,细胞膜上ASIC1和ASIC2a的表达没有野生型完整和连续,荧光强度减弱。
结论:
1. PICK1基因敲除后,培养的小鼠皮质神经元ASIC电流幅度减少,酸诱导的胞内钙增加也减少。因为在pH 6.0的实验条件下,主要是激活ASIC1a同聚体电流,而且在中枢神经元中,只有ASIC1a同聚体被激活后才对Ca2+通透,推测PICK1基因增加ASICs的功能主要是通过ASIC1a来实现的,ASIC2a也部分参与其中。
2. PICK1影响ASICs功能的机制并不在于PICK1敲除后ASIC1或ASIC2a的基因和蛋白表达水平减少,而可能是PICK1的缺失导致ASICs发生定位的改变,其蛋白从膜上转运到胞浆内所致,即ASICs的下膜作用。
第二部分PICK1基因敲除对蛋白激酶C调节ASICs功能的影响
目的:ASICs的调控受很多因素的影响,包括细胞外和细胞内因素。蛋白激酶PKC是常见的调节ASICs功能的上游因子之一,ASICs上有PKC的磷酸化位点。而PICK1又以其特定的PDZ区域与ASIC1a、ASIC2a和ASIC2b的C末端结合,故上述作用很多都与PICK1有关。既然在上文中我们已经证实,PICK1对ASICs功能的发挥起到重要作用,其基因的缺失会影响ASICs电流以及钙的通透性,那么,其进一步的机制是否与蛋白激酶C有关?PKC、PICK1和ASICs之间是一个怎样的关系和相互作用?这些都值得我们去进一步探讨。
方法:采用全细胞膜片钳、钙影像技术,western blotting和细胞免疫荧光技术,观察PICK1基因敲除前后,PKC激动剂和抑制剂对ASICs功能如何影响,探讨可能的机制。
结果:
1. PKC对小鼠皮质神经元ASICs电流有正性调节作用,该作用依赖于PICK1基因。在给予PKC激动剂或抑制剂前,ASICs电流的幅值在野生型小鼠神经元为228.60±20.09pA (n=13),纯合子为65.40±6.96pA (n=10)。当分别给予10μM GF109203X(PKC抑制剂)和1μM PMA(PKC激动剂)预孵育30 min后,野生型小鼠的电流幅值分别下降为107.50±15.75 pA (n=19, p<0.01 vs control),和增加为756.9±144.9 pA (n=11, p<0.01 vs control)。而在纯合子小鼠组,与对照组相比,无论是PKC抑制剂还是PKC激动剂对ASICs的影响均不明显,其电流值分别维持在64.4±4.81 pA (n=7, p>0.05 vs control )和84.3±13.05 pA (n=10, p>0.05 vs control)。
2. PKC对小鼠皮质神经元ASICs介导的钙升高有正性调节作用,该作用也依赖于PICK1的介导。细胞外酸性环境(pH=6.0)诱导出的瞬时钙升高,野生型对照组△F/F为0.662±0.062 (n=17),而纯合子为0.228±0.016 (n=33)。分别给予10μM GF109203X和1μM PMA预孵育后,野生型小鼠钙升高发生了明显改变,即GF109203X使△F/F下降至0.175±0.014 (n=26, p<0.01 vs control),PMA则使△F/F升高到1.08±0.112 (n=14, p<0.01 vs control)。而纯合子小鼠组与对照组相比,无论是PKC抑制剂还是PKC激动剂对△F/F的影响均不明显,其值分别维持在0.218±0.024 (n=20, p>0.05 vs control)和0.267±0.025 (n=13, p>0.05 vs control)。
3. PICK1基因敲除后,PKC的蛋白表达和分布不变。Western blotting和免疫荧光的结果均显示,小鼠皮质上无论是总PKC还是特异性与PICK1作用的PKCα的蛋白表达量都未发生改变;PKCα在细胞膜上和胞浆内均匀分布。
结论:
1. PKC对皮质神经元ASICs电流和ASICs介导的胞内钙增加的正性调节作用依赖于PICK1基因的存在,PICK1在其中起到了桥梁作用。
2. PICK1基因敲除后小鼠皮质上PKC和PKCα的蛋白表达总量和分布不变。进一步说明,PKC是通过PICK1的作用调节ASICs通道,PICK1敲除后,这一连接消失,而导致其调节ASICs的功能减弱。
第三部分PICK1基因敲除对蛋白激酶A调节ASICs功能的影响
目的:蛋白激酶PKA是除PKC外的另一常见的调节ASICs功能的上游因子,ASICs上也有PKA的磷酸化位点,能被其磷酸化,并与PICK1有关。PICK1基因的缺失会影响ASICs电流以及钙的释放,那么,其进一步的机制是否与蛋白激酶A也有关?PKA、PICK1和ASICs之间是一个怎样的关系和相互作用?这些也值得我们去进一步探讨。
方法:采用全细胞膜片钳、钙影像技术,western blotting和细胞免疫荧光技术,观察PICK1基因敲除前后,PKA激动剂和抑制剂对ASICs功能如何影响,探讨可能的机制。
结果:
1. PKA对小鼠皮质神经元ASICs电流及ASICs介导的胞内钙增加有负性调节作用。在给予PKA激动剂或抑制剂前,野生型ASICs电流幅值约为224.2±17.59 pA (n = 15),10μM forskolin(PKA激动剂)与神经元共同孵育30 min后,使电流幅值减小为108±14.67 pA (n=9, P<0.001 vs control),5μM PKA inhibitor fragment 6-22 amide(PKA抑制剂,PKAI)则增加ASICs电流,其值为306.3±22.9 pA (n=12, P<0.01 vs control)。酸诱导的钙升高结果,对照组△F/F值为0.708±0.07 (n=20),10μM forskolin使其减少为0.140±0.013 (n=17,p<0.001 vs control),而5μM PKAI使其增加为0.978±0.108 (n=19,p<0.05 vs control)。
2. PICK1基因敲除后,PKA对小鼠皮质神经元ASICs功能的负性调节作用减弱。用培养的PICK1-/-小鼠皮质神经元,观察forskolin和PKAI对ASICs电流的影响。对照组的电流幅值为61.2±9.62 pA (n=14),当分别与10μM forskolin和5μM PKAI共同孵育30 min后,仅forskolin使电流值减少约40%,为36.15±10.15pA (n=10, p<0.05 vs control),用PKAI后电流值仍然维持在和60.82±11.43 pA (n=11, p>0.05 vs control)。酸诱导皮质神经元钙内流增加的改变结果也与ASICs电流的变化一致,对照组△F/F值为0.195±0.02 (n=26),forskolin使其值降为0.157±0.011 (n=20,p<0.05 vs control),PKAI与神经元共同孵育30 min后,其值仍维持在0.170±0.0218 (n=28,p>0.05 vs control)。3. PICK1基因敲除后,皮质上PKA蛋白表达总量未发生改变。4. PKA对ASICs表达无明显影响,在加入forskolin和PKAI孵育24小时后,神经元总的荧光密度不变(p>0.05 vs control)。
结论:
1. PKA对培养的皮质神经元ASICs功能(ASIC电流和ASICs诱导的钙升高)产生负性调节作用;而PICK1基因敲除后,这种调节作用减弱。即PKA对ASICs功能的负性调节仍然依赖PICK1的作用,但并非全部,可能存在PKA的直接作用或其它影响因素。
2. PICK1基因敲除后小鼠皮质上PKA的蛋白表达总量不变,故并非PKA的数量影响了ASIC1和ASIC2a的功能。
3. PKA激动剂和抑制剂对野生型小鼠皮质神经元ASICs的表达无影响,故PKA的直接作用并未体现在对ASICs通道数量的改变,可能有其它因素。
总结:
1. PICK1基因敲除后,小鼠皮质神经元ASICs的功能减弱,其机制可能是ASICs定位发生改变,导致其在细胞膜上的分布减少所致。
2. PKC对皮质神经元ASICs的功能有正性调节作用,该作用的发挥依赖于PICK1基因。基因敲除后,正性调节的消失与PICK1的缺失有关,而不是PKC蛋白总量的改变。
3. PKA对皮质神经元ASICs的功能有负性调节作用,该作用的发挥部分依赖于PICK1基因的存在,可能还有自身直接作用或其它影响因素存在。基因敲除后,PKA蛋白表达总量不变,但PICK1作用的消失使PKA的负性调节作用受抑制。
PartⅠEffects of PICK1 gene knockout on the functions and expressions of ASICs
Aim: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 ASICs
A. 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 ASICs
A. 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 C
Aim: 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 A
Aim: 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.
引文
1. Ming Y, Zhang H, Long L, Wang F, Chen J and Zhen X. (2006) Modulation of Ca2+ signals by phosphatidylinositol-linked novel D1 dopamine receptor in hippocampal neurons. J Neurochemistry. 98:1316-1323
2. Hughes PA, Brierley SM, Young RL, Blackshaw LA. (2007) Localization and comparative analysis of acid-sensing ion channel (ASIC1, 2, and 3) mRNA expression in mouse colonic sensory neurons within thoracolumbar dorsal root ganglia. J. Comp. Neurol. 500(5): 863-875.
3. Gao J, Wu LJ, Xu L, Xu TL. (2004) Properties of the proton-evoked currents and their modulation by Ca2+ and Zn2+ in the acutely dissociated hippocampus CA1 neurons. Brain Res 1017: 197–207.
4. Chu XP, Wemmie JA, Wang WZ, Zhu XM, Saugstad JA, Price MP, Simon RP, Xiong ZG. (2004) Subunit-dependent high-affinity zinc inhibition of acid-sensing ion channels. J Neurosci 24: 8678–8689.
5. Wang W, Yu Y, Xu TL. (2007) Modulation of acid-sensing ion channels by Cu(2+) in cultured hypothalamic neurons of the rat. Neuroscience. 145(2): 631-41.
6. Askwith CC, Cheng C, Ikuma M, Benson C, Price MP, Welsh MJ. (2000) Neuropeptide FF and FMRFamide potentiate acid-evoked currents from sensory neurons and proton-gated DEG/ENaC channels. Neuron 26: 133–141.
7. Xie J, Price MP, Wemmie JA, et al. ASIC3 and ASIC1 mediate FMRFamide-related peptide enhancement of H+-gated currents in cultured dorsal root ganglion neurons. J Neurophysiol. 2003, 89:2459–2465.
8. Catarsi S, Babinski K, Seguela P. Selective modulation of heteromeric ASIC proton-gated channels by neuropeptide FF. Neuropharmacology. 2001, 41:592–600.
9. Duggan A, Garcia-Anoveros J, Corey DP. (2002) The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons. J. Biol. Chem. 277(7): 5203-5208.
10. Hruska-Hageman AM, Wemmie JA, Price MP, Welsh MJ. (2002) Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ionchannel). Biochem J. 361(Pt 3): 443-450
11. Price MP, Snyder PM and Welsh MJ. (1996) Cloning and expression of a novel human brain Na+ channel. J. Biom. Chem. 271: 7879-7882.
12. Akopian AN, Chen CC, Ding Y, Cesare P, Wood JN. (2000) A new member of the acid-sensing ion channel family. Neuroreport 11: 2217-2222.
13. Alvarez de la Rosa D, Zhang P, Shao D, White F, Canessa CM. (2002) Functional implications of the location and activity of acid-sensitive channels in rat peripheral nervous system. Proc. Natl. Acad. Sci. U. S. A. 99: 2326-2331.
14. Alvarez de la Rosa D, Krueger SR, Kolar A, Shao D, Fitzsimonds RM, Canessa CM. (2003) Distribution, subcellular localization and ontogeny of ASIC1 in the mammalian central nervous system. J Physiol. 546(Pt 1):77-87.
15. Waldmann R, Champigny G, Bassilana F, Heurteaux C and Lazdunski M. (1997) A proton-gated cation involved in acid-sensing. Nature 386: 173–177
16. Lingueglia E, Weille JR, Bassilana F, Heurteaux C, Sakai H, Waldmann R, Lazdunski M. (1997) A modulatory subunit of acid sensing ion channel in brain and dorsal root ganglion cells. J. Biom. Chem. 272: 29778-29783.
17. Xiong ZG, Zhu XM, Chu XP, et al. (2004) Neuroprotection in ischemia: blocking calcium permeable acid-sensing ion channels. Cell. 118 (6): 687–698.
18. Xia J, Chung HJ, Wihler C, Huganir RL, Linden DJ. Cerebellar long-term depression requires PKC-regulated interactions between GluR2/3 and PDZ domain-containing proteins. Neuron. 2000 Nov; 28(2):499-510
19. Xia J, Zhang X, Staudinger J, Huganir RL. Clustering of AMPA receptors by the synaptic PDZ domain-containing protein PICK1. Neuron. 1999 Jan; 22(1):179-87.
20. Torres GE, Yao WD, Mohn AR, Quan H, Kim KM, Levey AI, Staudinger J and Caron MG. (2001) Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domain-containing protein PICK1. Neuron 30: 121-134.
21. Torres R, Firestein BL, Dong H, Staudinger J, Olson EN, Huganir, RL, Bredt DS, GaleNW and Yancopoulos GD. (1998) PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands. Neuron 21: 1453-1463.
22. Xu J, Xia J. (2007) Structure and Function of PICK1. Neurosignals. 15(4):190-201.
23. Boudin H, Doan A, Xia J, Shigemoto R, Huganir RL, Worley P and Craig AM. (2000) Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site. Neuron 28: 485-497.
24. Dev KK, Nishimune A, Henley JM and Nakanishi S. (1999) The protein kinase C alpha binding protein PICK1 interacts with short but not long form alternative splice variants of AMPA receptor subunits. Neuropharmacology 38: 635-644.
1. Staudinger J, Zhou J, Burgess R, Elledge SJ, Olson EN. (1995) PICK1: a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system. J. Cell. Biol. 128: 263–271.
2. Masukawa K, Sakai N, Ohiho S, Shirai Y and Saito N. (2006). Spatiotemporal analysis of the molecular interaction between PICK1 and PKC. Acta Histochem. Cytochem. 39(6): 173-181.
3. Perez J, Khatri L, Chang C, Srivastava S, Osten P and Ziff EB. (2001) PICK1 targets protein kinase Calpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surface levels of the AMPA-type glutamate receptor subunit 2. J. Neurosci. 21: 5417-5428.
4. Baron A, Deval E, Salinas M, Lingueglia E, Voilley N, Lazdunski M. (2002) Protein kinase C stimulates the acid-sensing ion channel ASIC2a via the PDZ domain-containing protein PICK1. J Biol Chem. 277(52): 50463-50468.
5. Deval E, Salinas M, Baron A, et al. (2004) ASIC2b-dependent Regulation of ASIC3, an Essential Acid-sensing Ion Channel Subunit in Sensory Neurons via the Partner Protein PICK-1. J. Biol. Chem. 279(19): 19531–19539.
6. Price MP, Snyder PM and Welsh MJ. (1996) Cloning and expression of a novel human brain Na+ channel. J. Biom. Chem. 271: 7879-7882.
7. Waldmann R, Champigny G, Bassilana F, Heurteaux C and Lazdunski M. (1997) A proton-gated cation involved in acid-sensing. Nature 386: 173–177
8. Lingueglia E, Weille JR, Bassilana F, Heurteaux C, Sakai H, Waldmann R, Lazdunski M. (1997) A modulatory subunit of acid sensing ion channel in brain and dorsal root ganglion cells. J. Biom. Chem. 272: 29778-29783.
9. Xiong ZG, Zhu XM, Chu XP, et al. (2004) Neuroprotection in ischemia: blocking calcium permeable acid-sensing ion channels. Cell. 118 (6): 687–698
10. Berdiev BK, Xia J, Jovov B, Markert JM, Mapstone TB, Gillespie GY, Fuller CM,Bubien JK, Benos DJ. (2002) Protein kinase C isoform antagonism controls BNaC2 (ASIC1) function. J Biol Chem. 277(48): 45734-45740.
1. Leonard AS, Yermolaieva O, Hruska-Hageman A, et al. (2003) cAMP-dependent protein kinase phosphorylation of the acid-sensing ion channel-1 regulates its binding to the protein interacting with C-kinase-1. Proc. Natl. Acad. Sci. USA. 100: 2029-2034
2. Masukawa K, Sakai N, Ohiho S, Shirai Y and Saito N. (2006). Spatiotemporal analysis of the molecular interaction between PICK1 and PKC. Acta Histochem. Cytochem. 39(6): 173-181.
3. Deval E, Salinas M, Baron A, et al. ASIC2b-dependent Regulation of ASIC3, an Essential Acid-sensing Ion Channel Subunit in Sensory Neurons via the Partner Protein PICK-1. J. Biol. Chem. 2004, 279(19): 19531–19539.
4. Hruska-Hageman AM, Wemmie JA, Price MP, Welsh MJ. 2002. Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel). Biochem J. 361(Pt 3): 443-450
5. Duggan A, Garcia-Anoveros J, Corey DP. The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons. J. Biol. Chem. 2002. 277(7): 5203-5208.
6. Bassilana F, Champigny G, Waldmann R, et al. (1997) The acid-sensitive ionic channel subunit ASIC and the mammalian degenerin MDEG form a heteromultimeric H+-gated Na+ channel with novel properties. J. Biol. Chem. 272 (46): 28819 - 28822.
1. Krishtal OA, Pidoplichko V. A receptor for protons in the nerve cell membrane. Neuroscience. 1980, 5 (12): 2325 -2327.
2. Waldmann R, Champigny G, Bassilana F, et al. A proton-gated cation channel involved in acid-sensing. Nature. 1997, 386(6621): 173 -177.
3. Krishtal OA. The ASICs: signaling molecules? Modulators? Trends Neurosci 2003, 26 (9) : 477 - 483.
4. Alvarez de la Rosa D, Krueger SR, Kolar A, et al. Distribution, subcellular localization and ontogeny of ASIC1 in the mammalian central nervous system. J. Physiol. 2003, 546: 77–87.
5. Gunthorpe MJ, Smith GD, Davis JB, et al. Characterisation of a human acid-sensing ion channel (hASIC1a) endogenously expressed in HEK293 cells. Pflugers Arch 2001, 442 (5) : 668- 674.
6. Weng XC, Zheng JQ, Li J, et al. Underlying mechanism of ASIC1a involved in acidosis-induced cytotoxicity in rat C6 glioma cells. Acta Pharmacol Sin. 2007, 28(11):1731-1736.
7. Lingueglia, E, Weille, JR, Bassilana, F, et al. A modulatory subunit of acid sensing ion channels in brain and dorsal root ganglion cells. J. Biol. Chem. 1997, 272: 29778–29783.
8. Sutherland, SP, Benson, CJ, Adelman, JP, et al. Acid-sensing ion channel 3 matches the acid-gated current in cardiac ischemia-sensing neurons. Proc. Natl. Acad. Sci. USA 2001, 98: 711–716.
9. Waldmann, R, Bassilana, F, Weille, J, et al. Molecular cloning of a non-inactivating proton-gated Na+ channel specific for sensory neurons. J. Biol. Chem. 1997, 272: 20975–20978.
10. Akopian AN, Chen CC, Ding Y, et al. A new member of the acid-sensing ion channel family. Neuroreport 2000, 11: 2217-2222.
11. Grunder S, Geissler HS, Bassler EL, et al. A new member of acid-sensing ion channels from pituitary gland. Neuroreport 2000, 11:1607-1611.
12. Ye JH, Gao J, Wu YN, et al. Identification of acid-sensing ion channels in adenoid cystic carcinomas. Biochem Biophys Res Commun. 2007, 355(4): 986-992.
13. Ugawa S, Inagaki A, Yamamura H, et al. Acid-sensing ion channel-1b in the stereocilia of mammalian cochlear hair cells. Neuroreport. 2006, 17(12):1235-1239.
13. Jahr H, van Driel M, van Osch GJ, et al. Identification of acid-sensing ion channels in bone. Biochem Biophys Res Commun. 2005, 337(1): 349-354.
14. Ettaiche M, Guy N, Hofman P, et al. Acid-sensing ion channel 2 is important for retinal function and protects against light-induced retinal degeneration. J Neurosci. 2004, 24(5): 1005-1012.
16. Yiangou Y, Facer P, Smith JA, et al. Increased acid-sensing ion channel ASIC-3 in inflamed human intestine. Eur J Gastroenterol Hepatol. 2001, 13(8): 891-896.
17. Wemmie JA, Chen J, Askwith CC, et al. The acid-activated ion channel ASIC contributes to synaptic plasticity, learning and memory. Neuron. 2002, 34 (3): 463- 477.
15. 18. Wemmie JA, Coryell MW, Askwith CC, et al. Overexpression of acid-sensing ion channel 1a in transgenic mice increases acquired fear-related behavior. Proc. Natl. Acad. Sci. USA 2004, 101(10): 3621-3626.
16. 19. Wemmie JA, Askwith CC, Lamani E, et al. Acid-sensing ion channel 1 is localized in brain regions with high synaptic density and contributes to fear conditioning. J Neurosci. 2003, 23(13): 5496 - 5502.
17. 20. Ettaiche M, Deval E, Cougnon M, et al. Silencing acid-sensing ion channel 1a alters cone-mediated retinal function. J Neurosci 2006, 26 (21) : 5800– 5809.
18. 21. Ugawa S, Ueda T, Ishida Y, et al. Amiloride blockable acid-sensing ion channels are leading acid sensors expressed in human nociceptors. J Clin Invest. 2002, 110(8) :1185 - 1190.
19. 22. Jones NG, Slater R, Cadiou H, et al. Acid-induced pain and its modulation in humans. J Neurosci. 2004, 24 (48) : 10974-10979.
20. 23. Sluka KA, Price MP, Breese NM, et al. Chronic hyperalgesia induced by repeated acid injections in muscle is abolished by the loss of ASIC3 , but not ASIC1. Pain. 2003, 106(3):229 - 239.
21. Chen CC, Zimmer A, Sun WH, et al. A role for ASIC3 in the modulation of high-intensity pain stimuli. Proc Natl Acad Sci USA. 2002, 99(13): 8992– 8997.
22. Babinski K, Le KT , Seguela P. Molecular cloning and regional distribution of a human proton receptor subunit with biphasic functional properties. J Neurochem. 1999, 72(1):51– 57.
23. Price MP, Thompson RJ, Eshcol JO, et al. Stomatin modulates gating of acid-sensing ion channels. J. Biol. Chem. 2004, 279(51): 53886– 53891.
24. Drew LJ, Rohrer DK, Price MP, et al. Acid-sensing ion channels ASIC2 and ASIC3 do not contribute to mechanically activated currents in mammalian sensory neurones. J Physiol. 2004, 556(Pt 3): 691 - 710.
25. Roza C, Puel JL, Kress M, et al. Knockout of the ASIC2 channel in mice does not impair cutaneous mechanosensation, visceral mechanonociception and hearing. J Physiol. 2004, 558 (Pt 2): 659 -669.
26. Richter TA, Dvoryanchikov GA, Roper SD, et al. Acid-sensing ion channel 2 is notnecessary for sour taste in mice. J Neurosc., 2004, 24 (16): 4088 - 4091.
27. EttaicheM, Guy N, Hofman P, et al. Acid-sensing ion channel 2 is important for retinal function and protects against light-induced retinal degeneration. J Neurosci. 2004, 24 (5): 1005 - 1012.
28. Xiong ZG, Zhu XM, Chu XP, et al. Neuroprotection in ischemia: blocking calcium permeable acid-sensing ion channels. Cell. 2004, 118 (6): 687–698.
29. Gao J, Duan B, Wang DG, et al. Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death. Neuron. 2005, 48 (4): 635 - 646.
30. Naves LA, McCleskey EW. An acid-sensing ion channel that detects ischemic pain. Braz J Med Biol Res. 2005, 38(11): 1561-1569.
31. Immke DC, McCleskey EW. Lactate enhances the acid-sensing Na+ channel on ischemia-sensing neurons. Nat Neurosci. 2001, 4(9): 869-870.
32. Yermolaieva, O, Leonard, AS, Schnizler, MK, et al. Extracellular acidosis increases neuronal cell calcium by activating acid-sensing ion channel 1a. Proc. Natl. Acad. Sci. USA 2004, 101: 6752–6757.
33. Chen, CC, England, S, Akopian, AN, et al. A sensory neuron-specific, proton-gated ion channel. Proc. Natl. Acad. Sci. USA 1998, 95: 10240–10245.
34. Askwith CC, Cheng C, Ikuma M, et al. Neuropeptide FF and FMRFamide potentiate acidevoked currents from sensory neurons and proton-gated DEG/ENaC channels. Neuron. 2000, 26:133–141.
35. Xie J, Price MP, Wemmie JA, et al. ASIC3 and ASIC1 mediate FMRFamide-related peptide enhancement of H+-gated currents in cultured dorsal root ganglion neurons. J Neurophysiol. 2003, 89:2459–2465.
36. Catarsi S, Babinski K, Seguela P. Selective modulation of heteromeric ASIC proton-gated channels by neuropeptide FF. Neuropharmacology. 2001, 41:592–600.
37. Askwith CC, Wemmie JA, Price MP, et al. Acid-sensing ion channel 2 (ASIC2) modulates ASIC1 H+-activated currents in hippocampal neurons. J. Biol. Chem. 2004,279:18296–18305.
38. Deval E, Baron A, Lingueglia E, et al. Effects of neuropeptide SF and related peptides on acid sensing ion channel 3 and sensory neuron excitability. Neuropharmacology. 2003, 44:662–671.
39. Chu XP, Wemmie JA, Wang WZ, et al. Subunit-dependent high-affinity zinc inhibition of acid-sensing ion channels. J N eurosci. 2004, 24 (40): 8678 - 8689.
40. Baron A, Schaefer L, Lingueglia E, et al. Zn2+ and H+ are coactivators of acid-sensing ion channels. J. Biol. Chem., 2001, 276(38): 35361 -35367.
41. Gao J, Wu LJ, Xu L, et al. Properties of the proton-evoked currents and their modulation by Ca2+ and Zn2+ in the acutely dissociated hippocampus CA1 neurons. Brain Res. 2004, 1017(1-2):197-207.
42. Immke DC, McCleskey EW. Protons open acid-sensing ion channels by catalyzing relief of Ca2+ blockade. Neuron. 2003, 37(1): 75 - 84.
43. Paukert M, Babini E, Pusch M, et al. Identification of the Ca2+ blocking site of acid-sensing ion channel (ASIC) 1: Implications for channel gating. J Gen Physiol. 2004, 124 (4): 383 -394.
44. Zhang P, Canessa CM. Single channel properties of rat acid-sensitive ion channel-1α, -2a , and -3 expressed in Xenopus oocytes. J. Gen. Physiol. 2002, 120 (4):553 - 566.
45. Xu TL, Xing ZG. Dynamic Regulation of Acid-Sensing Ion Channels by Extracellular and Intracellular Modulators. Current Medicinal Chemistry. 2007, 14: 1753-1763
46. Andrey F, Tsintsadze T, Volkova T, et al. Acid sensing ionic channels: modulation by redox reagents. Biochim. Biophys. Acta. 2005, 1745(1): 1-6.
47. Chu XP, Close N, Saugstad JA, et al. ASIC1a-specific modulation of acid-sensing ion channels in mouse cortical neurons by redox reagents. J Neurosci. 2006, 26 (20): 5329 - 5339.
48. Baron A, Deval E, Salinas M, Lingueglia E, Voilley N, Lazdunski M. Protein kinase C stimulates the acid-sensing ion channel ASIC2a via the PDZ domain-containing proteinPICK1. J. Biol. Chem. 2002, 277(52): 50463-50468.
49. Deval E, Salinas M, Baron A, et al. ASIC2b-dependent Regulation of ASIC3, an Essential Acid-sensing Ion Channel Subunit in Sensory Neurons via the Partner Protein PICK-1. J. Biol. Chem. 2004, 279(19): 19531–19539.
50. Bassilana F, Champigny G, Waldmann R, et al. The acid-sensitive ionic channel subunit ASIC and the mammalian degenerin MDEG form a heteromultimeric H+-gated Na+ channel with novel properties. J. Biol. Chem. 1997, 272 (46): 28819 - 28822.
51. Leonard AS, Yermolaieva O, Hruska-Hageman A, et al. cAMP-dependent protein kinase phosphorylation of the acid-sensing ion channel-1 regulates its binding to the protein interacting with C-kinase-1. Proc. Natl. Acad. Sci. USA. 2003, 100: 2029-2034
52. Hruska-Hageman AM, Wemmie JA, Price MP, Welsh MJ. 2002. Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel). Biochem J. 361(Pt 3): 443-450
53. Duggan A, Garcia-Anoveros J, Corey DP. The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons. J. Biol. Chem. 2002. 277(7): 5203-5208.
54. Anzai N, Deval E, Schaefer L, et al. The multivalent PDZ domain-containing protein CIPP is a partner of acid-sensing ion channel 3 in sensory neurons. J. Biol. Chem. 2002, 277(19): 16655-16661.
55. Yudin YK, Tamarova ZA, Ostrovskaya OI, Moroz LL, Krishtal OA. RFa-related peptides are algogenic: evidence in vitro and in vivo. Eur J Meurosci. 2004, 20(5):1419-1423.
56. Jasi J, Furukawa H, Gonzales EB, Gouaux E. Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH. Nature. 2007, 449(7160): 316-323.
2. Hughes PA, Brierley SM, Young RL, Blackshaw LA. (2007) Localization and comparative analysis of acid-sensing ion channel (ASIC1, 2, and 3) mRNA expression in mouse colonic sensory neurons within thoracolumbar dorsal root ganglia. J. Comp. Neurol. 500(5): 863-875.
3. Gao J, Wu LJ, Xu L, Xu TL. (2004) Properties of the proton-evoked currents and their modulation by Ca2+ and Zn2+ in the acutely dissociated hippocampus CA1 neurons. Brain Res 1017: 197–207.
4. Chu XP, Wemmie JA, Wang WZ, Zhu XM, Saugstad JA, Price MP, Simon RP, Xiong ZG. (2004) Subunit-dependent high-affinity zinc inhibition of acid-sensing ion channels. J Neurosci 24: 8678–8689.
5. Wang W, Yu Y, Xu TL. (2007) Modulation of acid-sensing ion channels by Cu(2+) in cultured hypothalamic neurons of the rat. Neuroscience. 145(2): 631-41.
6. Askwith CC, Cheng C, Ikuma M, Benson C, Price MP, Welsh MJ. (2000) Neuropeptide FF and FMRFamide potentiate acid-evoked currents from sensory neurons and proton-gated DEG/ENaC channels. Neuron 26: 133–141.
7. Xie J, Price MP, Wemmie JA, et al. ASIC3 and ASIC1 mediate FMRFamide-related peptide enhancement of H+-gated currents in cultured dorsal root ganglion neurons. J Neurophysiol. 2003, 89:2459–2465.
8. Catarsi S, Babinski K, Seguela P. Selective modulation of heteromeric ASIC proton-gated channels by neuropeptide FF. Neuropharmacology. 2001, 41:592–600.
9. Duggan A, Garcia-Anoveros J, Corey DP. (2002) The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons. J. Biol. Chem. 277(7): 5203-5208.
10. Hruska-Hageman AM, Wemmie JA, Price MP, Welsh MJ. (2002) Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ionchannel). Biochem J. 361(Pt 3): 443-450
11. Price MP, Snyder PM and Welsh MJ. (1996) Cloning and expression of a novel human brain Na+ channel. J. Biom. Chem. 271: 7879-7882.
12. Akopian AN, Chen CC, Ding Y, Cesare P, Wood JN. (2000) A new member of the acid-sensing ion channel family. Neuroreport 11: 2217-2222.
13. Alvarez de la Rosa D, Zhang P, Shao D, White F, Canessa CM. (2002) Functional implications of the location and activity of acid-sensitive channels in rat peripheral nervous system. Proc. Natl. Acad. Sci. U. S. A. 99: 2326-2331.
14. Alvarez de la Rosa D, Krueger SR, Kolar A, Shao D, Fitzsimonds RM, Canessa CM. (2003) Distribution, subcellular localization and ontogeny of ASIC1 in the mammalian central nervous system. J Physiol. 546(Pt 1):77-87.
15. Waldmann R, Champigny G, Bassilana F, Heurteaux C and Lazdunski M. (1997) A proton-gated cation involved in acid-sensing. Nature 386: 173–177
16. Lingueglia E, Weille JR, Bassilana F, Heurteaux C, Sakai H, Waldmann R, Lazdunski M. (1997) A modulatory subunit of acid sensing ion channel in brain and dorsal root ganglion cells. J. Biom. Chem. 272: 29778-29783.
17. Xiong ZG, Zhu XM, Chu XP, et al. (2004) Neuroprotection in ischemia: blocking calcium permeable acid-sensing ion channels. Cell. 118 (6): 687–698.
18. Xia J, Chung HJ, Wihler C, Huganir RL, Linden DJ. Cerebellar long-term depression requires PKC-regulated interactions between GluR2/3 and PDZ domain-containing proteins. Neuron. 2000 Nov; 28(2):499-510
19. Xia J, Zhang X, Staudinger J, Huganir RL. Clustering of AMPA receptors by the synaptic PDZ domain-containing protein PICK1. Neuron. 1999 Jan; 22(1):179-87.
20. Torres GE, Yao WD, Mohn AR, Quan H, Kim KM, Levey AI, Staudinger J and Caron MG. (2001) Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domain-containing protein PICK1. Neuron 30: 121-134.
21. Torres R, Firestein BL, Dong H, Staudinger J, Olson EN, Huganir, RL, Bredt DS, GaleNW and Yancopoulos GD. (1998) PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands. Neuron 21: 1453-1463.
22. Xu J, Xia J. (2007) Structure and Function of PICK1. Neurosignals. 15(4):190-201.
23. Boudin H, Doan A, Xia J, Shigemoto R, Huganir RL, Worley P and Craig AM. (2000) Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site. Neuron 28: 485-497.
24. Dev KK, Nishimune A, Henley JM and Nakanishi S. (1999) The protein kinase C alpha binding protein PICK1 interacts with short but not long form alternative splice variants of AMPA receptor subunits. Neuropharmacology 38: 635-644.
1. Staudinger J, Zhou J, Burgess R, Elledge SJ, Olson EN. (1995) PICK1: a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system. J. Cell. Biol. 128: 263–271.
2. Masukawa K, Sakai N, Ohiho S, Shirai Y and Saito N. (2006). Spatiotemporal analysis of the molecular interaction between PICK1 and PKC. Acta Histochem. Cytochem. 39(6): 173-181.
3. Perez J, Khatri L, Chang C, Srivastava S, Osten P and Ziff EB. (2001) PICK1 targets protein kinase Calpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surface levels of the AMPA-type glutamate receptor subunit 2. J. Neurosci. 21: 5417-5428.
4. Baron A, Deval E, Salinas M, Lingueglia E, Voilley N, Lazdunski M. (2002) Protein kinase C stimulates the acid-sensing ion channel ASIC2a via the PDZ domain-containing protein PICK1. J Biol Chem. 277(52): 50463-50468.
5. Deval E, Salinas M, Baron A, et al. (2004) ASIC2b-dependent Regulation of ASIC3, an Essential Acid-sensing Ion Channel Subunit in Sensory Neurons via the Partner Protein PICK-1. J. Biol. Chem. 279(19): 19531–19539.
6. Price MP, Snyder PM and Welsh MJ. (1996) Cloning and expression of a novel human brain Na+ channel. J. Biom. Chem. 271: 7879-7882.
7. Waldmann R, Champigny G, Bassilana F, Heurteaux C and Lazdunski M. (1997) A proton-gated cation involved in acid-sensing. Nature 386: 173–177
8. Lingueglia E, Weille JR, Bassilana F, Heurteaux C, Sakai H, Waldmann R, Lazdunski M. (1997) A modulatory subunit of acid sensing ion channel in brain and dorsal root ganglion cells. J. Biom. Chem. 272: 29778-29783.
9. Xiong ZG, Zhu XM, Chu XP, et al. (2004) Neuroprotection in ischemia: blocking calcium permeable acid-sensing ion channels. Cell. 118 (6): 687–698
10. Berdiev BK, Xia J, Jovov B, Markert JM, Mapstone TB, Gillespie GY, Fuller CM,Bubien JK, Benos DJ. (2002) Protein kinase C isoform antagonism controls BNaC2 (ASIC1) function. J Biol Chem. 277(48): 45734-45740.
1. Leonard AS, Yermolaieva O, Hruska-Hageman A, et al. (2003) cAMP-dependent protein kinase phosphorylation of the acid-sensing ion channel-1 regulates its binding to the protein interacting with C-kinase-1. Proc. Natl. Acad. Sci. USA. 100: 2029-2034
2. Masukawa K, Sakai N, Ohiho S, Shirai Y and Saito N. (2006). Spatiotemporal analysis of the molecular interaction between PICK1 and PKC. Acta Histochem. Cytochem. 39(6): 173-181.
3. Deval E, Salinas M, Baron A, et al. ASIC2b-dependent Regulation of ASIC3, an Essential Acid-sensing Ion Channel Subunit in Sensory Neurons via the Partner Protein PICK-1. J. Biol. Chem. 2004, 279(19): 19531–19539.
4. Hruska-Hageman AM, Wemmie JA, Price MP, Welsh MJ. 2002. Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel). Biochem J. 361(Pt 3): 443-450
5. Duggan A, Garcia-Anoveros J, Corey DP. The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons. J. Biol. Chem. 2002. 277(7): 5203-5208.
6. Bassilana F, Champigny G, Waldmann R, et al. (1997) The acid-sensitive ionic channel subunit ASIC and the mammalian degenerin MDEG form a heteromultimeric H+-gated Na+ channel with novel properties. J. Biol. Chem. 272 (46): 28819 - 28822.
1. Krishtal OA, Pidoplichko V. A receptor for protons in the nerve cell membrane. Neuroscience. 1980, 5 (12): 2325 -2327.
2. Waldmann R, Champigny G, Bassilana F, et al. A proton-gated cation channel involved in acid-sensing. Nature. 1997, 386(6621): 173 -177.
3. Krishtal OA. The ASICs: signaling molecules? Modulators? Trends Neurosci 2003, 26 (9) : 477 - 483.
4. Alvarez de la Rosa D, Krueger SR, Kolar A, et al. Distribution, subcellular localization and ontogeny of ASIC1 in the mammalian central nervous system. J. Physiol. 2003, 546: 77–87.
5. Gunthorpe MJ, Smith GD, Davis JB, et al. Characterisation of a human acid-sensing ion channel (hASIC1a) endogenously expressed in HEK293 cells. Pflugers Arch 2001, 442 (5) : 668- 674.
6. Weng XC, Zheng JQ, Li J, et al. Underlying mechanism of ASIC1a involved in acidosis-induced cytotoxicity in rat C6 glioma cells. Acta Pharmacol Sin. 2007, 28(11):1731-1736.
7. Lingueglia, E, Weille, JR, Bassilana, F, et al. A modulatory subunit of acid sensing ion channels in brain and dorsal root ganglion cells. J. Biol. Chem. 1997, 272: 29778–29783.
8. Sutherland, SP, Benson, CJ, Adelman, JP, et al. Acid-sensing ion channel 3 matches the acid-gated current in cardiac ischemia-sensing neurons. Proc. Natl. Acad. Sci. USA 2001, 98: 711–716.
9. Waldmann, R, Bassilana, F, Weille, J, et al. Molecular cloning of a non-inactivating proton-gated Na+ channel specific for sensory neurons. J. Biol. Chem. 1997, 272: 20975–20978.
10. Akopian AN, Chen CC, Ding Y, et al. A new member of the acid-sensing ion channel family. Neuroreport 2000, 11: 2217-2222.
11. Grunder S, Geissler HS, Bassler EL, et al. A new member of acid-sensing ion channels from pituitary gland. Neuroreport 2000, 11:1607-1611.
12. Ye JH, Gao J, Wu YN, et al. Identification of acid-sensing ion channels in adenoid cystic carcinomas. Biochem Biophys Res Commun. 2007, 355(4): 986-992.
13. Ugawa S, Inagaki A, Yamamura H, et al. Acid-sensing ion channel-1b in the stereocilia of mammalian cochlear hair cells. Neuroreport. 2006, 17(12):1235-1239.
13. Jahr H, van Driel M, van Osch GJ, et al. Identification of acid-sensing ion channels in bone. Biochem Biophys Res Commun. 2005, 337(1): 349-354.
14. Ettaiche M, Guy N, Hofman P, et al. Acid-sensing ion channel 2 is important for retinal function and protects against light-induced retinal degeneration. J Neurosci. 2004, 24(5): 1005-1012.
16. Yiangou Y, Facer P, Smith JA, et al. Increased acid-sensing ion channel ASIC-3 in inflamed human intestine. Eur J Gastroenterol Hepatol. 2001, 13(8): 891-896.
17. Wemmie JA, Chen J, Askwith CC, et al. The acid-activated ion channel ASIC contributes to synaptic plasticity, learning and memory. Neuron. 2002, 34 (3): 463- 477.
15. 18. Wemmie JA, Coryell MW, Askwith CC, et al. Overexpression of acid-sensing ion channel 1a in transgenic mice increases acquired fear-related behavior. Proc. Natl. Acad. Sci. USA 2004, 101(10): 3621-3626.
16. 19. Wemmie JA, Askwith CC, Lamani E, et al. Acid-sensing ion channel 1 is localized in brain regions with high synaptic density and contributes to fear conditioning. J Neurosci. 2003, 23(13): 5496 - 5502.
17. 20. Ettaiche M, Deval E, Cougnon M, et al. Silencing acid-sensing ion channel 1a alters cone-mediated retinal function. J Neurosci 2006, 26 (21) : 5800– 5809.
18. 21. Ugawa S, Ueda T, Ishida Y, et al. Amiloride blockable acid-sensing ion channels are leading acid sensors expressed in human nociceptors. J Clin Invest. 2002, 110(8) :1185 - 1190.
19. 22. Jones NG, Slater R, Cadiou H, et al. Acid-induced pain and its modulation in humans. J Neurosci. 2004, 24 (48) : 10974-10979.
20. 23. Sluka KA, Price MP, Breese NM, et al. Chronic hyperalgesia induced by repeated acid injections in muscle is abolished by the loss of ASIC3 , but not ASIC1. Pain. 2003, 106(3):229 - 239.
21. Chen CC, Zimmer A, Sun WH, et al. A role for ASIC3 in the modulation of high-intensity pain stimuli. Proc Natl Acad Sci USA. 2002, 99(13): 8992– 8997.
22. Babinski K, Le KT , Seguela P. Molecular cloning and regional distribution of a human proton receptor subunit with biphasic functional properties. J Neurochem. 1999, 72(1):51– 57.
23. Price MP, Thompson RJ, Eshcol JO, et al. Stomatin modulates gating of acid-sensing ion channels. J. Biol. Chem. 2004, 279(51): 53886– 53891.
24. Drew LJ, Rohrer DK, Price MP, et al. Acid-sensing ion channels ASIC2 and ASIC3 do not contribute to mechanically activated currents in mammalian sensory neurones. J Physiol. 2004, 556(Pt 3): 691 - 710.
25. Roza C, Puel JL, Kress M, et al. Knockout of the ASIC2 channel in mice does not impair cutaneous mechanosensation, visceral mechanonociception and hearing. J Physiol. 2004, 558 (Pt 2): 659 -669.
26. Richter TA, Dvoryanchikov GA, Roper SD, et al. Acid-sensing ion channel 2 is notnecessary for sour taste in mice. J Neurosc., 2004, 24 (16): 4088 - 4091.
27. EttaicheM, Guy N, Hofman P, et al. Acid-sensing ion channel 2 is important for retinal function and protects against light-induced retinal degeneration. J Neurosci. 2004, 24 (5): 1005 - 1012.
28. Xiong ZG, Zhu XM, Chu XP, et al. Neuroprotection in ischemia: blocking calcium permeable acid-sensing ion channels. Cell. 2004, 118 (6): 687–698.
29. Gao J, Duan B, Wang DG, et al. Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death. Neuron. 2005, 48 (4): 635 - 646.
30. Naves LA, McCleskey EW. An acid-sensing ion channel that detects ischemic pain. Braz J Med Biol Res. 2005, 38(11): 1561-1569.
31. Immke DC, McCleskey EW. Lactate enhances the acid-sensing Na+ channel on ischemia-sensing neurons. Nat Neurosci. 2001, 4(9): 869-870.
32. Yermolaieva, O, Leonard, AS, Schnizler, MK, et al. Extracellular acidosis increases neuronal cell calcium by activating acid-sensing ion channel 1a. Proc. Natl. Acad. Sci. USA 2004, 101: 6752–6757.
33. Chen, CC, England, S, Akopian, AN, et al. A sensory neuron-specific, proton-gated ion channel. Proc. Natl. Acad. Sci. USA 1998, 95: 10240–10245.
34. Askwith CC, Cheng C, Ikuma M, et al. Neuropeptide FF and FMRFamide potentiate acidevoked currents from sensory neurons and proton-gated DEG/ENaC channels. Neuron. 2000, 26:133–141.
35. Xie J, Price MP, Wemmie JA, et al. ASIC3 and ASIC1 mediate FMRFamide-related peptide enhancement of H+-gated currents in cultured dorsal root ganglion neurons. J Neurophysiol. 2003, 89:2459–2465.
36. Catarsi S, Babinski K, Seguela P. Selective modulation of heteromeric ASIC proton-gated channels by neuropeptide FF. Neuropharmacology. 2001, 41:592–600.
37. Askwith CC, Wemmie JA, Price MP, et al. Acid-sensing ion channel 2 (ASIC2) modulates ASIC1 H+-activated currents in hippocampal neurons. J. Biol. Chem. 2004,279:18296–18305.
38. Deval E, Baron A, Lingueglia E, et al. Effects of neuropeptide SF and related peptides on acid sensing ion channel 3 and sensory neuron excitability. Neuropharmacology. 2003, 44:662–671.
39. Chu XP, Wemmie JA, Wang WZ, et al. Subunit-dependent high-affinity zinc inhibition of acid-sensing ion channels. J N eurosci. 2004, 24 (40): 8678 - 8689.
40. Baron A, Schaefer L, Lingueglia E, et al. Zn2+ and H+ are coactivators of acid-sensing ion channels. J. Biol. Chem., 2001, 276(38): 35361 -35367.
41. Gao J, Wu LJ, Xu L, et al. Properties of the proton-evoked currents and their modulation by Ca2+ and Zn2+ in the acutely dissociated hippocampus CA1 neurons. Brain Res. 2004, 1017(1-2):197-207.
42. Immke DC, McCleskey EW. Protons open acid-sensing ion channels by catalyzing relief of Ca2+ blockade. Neuron. 2003, 37(1): 75 - 84.
43. Paukert M, Babini E, Pusch M, et al. Identification of the Ca2+ blocking site of acid-sensing ion channel (ASIC) 1: Implications for channel gating. J Gen Physiol. 2004, 124 (4): 383 -394.
44. Zhang P, Canessa CM. Single channel properties of rat acid-sensitive ion channel-1α, -2a , and -3 expressed in Xenopus oocytes. J. Gen. Physiol. 2002, 120 (4):553 - 566.
45. Xu TL, Xing ZG. Dynamic Regulation of Acid-Sensing Ion Channels by Extracellular and Intracellular Modulators. Current Medicinal Chemistry. 2007, 14: 1753-1763
46. Andrey F, Tsintsadze T, Volkova T, et al. Acid sensing ionic channels: modulation by redox reagents. Biochim. Biophys. Acta. 2005, 1745(1): 1-6.
47. Chu XP, Close N, Saugstad JA, et al. ASIC1a-specific modulation of acid-sensing ion channels in mouse cortical neurons by redox reagents. J Neurosci. 2006, 26 (20): 5329 - 5339.
48. Baron A, Deval E, Salinas M, Lingueglia E, Voilley N, Lazdunski M. Protein kinase C stimulates the acid-sensing ion channel ASIC2a via the PDZ domain-containing proteinPICK1. J. Biol. Chem. 2002, 277(52): 50463-50468.
49. Deval E, Salinas M, Baron A, et al. ASIC2b-dependent Regulation of ASIC3, an Essential Acid-sensing Ion Channel Subunit in Sensory Neurons via the Partner Protein PICK-1. J. Biol. Chem. 2004, 279(19): 19531–19539.
50. Bassilana F, Champigny G, Waldmann R, et al. The acid-sensitive ionic channel subunit ASIC and the mammalian degenerin MDEG form a heteromultimeric H+-gated Na+ channel with novel properties. J. Biol. Chem. 1997, 272 (46): 28819 - 28822.
51. Leonard AS, Yermolaieva O, Hruska-Hageman A, et al. cAMP-dependent protein kinase phosphorylation of the acid-sensing ion channel-1 regulates its binding to the protein interacting with C-kinase-1. Proc. Natl. Acad. Sci. USA. 2003, 100: 2029-2034
52. Hruska-Hageman AM, Wemmie JA, Price MP, Welsh MJ. 2002. Interaction of the synaptic protein PICK1 (protein interacting with C kinase 1) with the non-voltage gated sodium channels BNC1 (brain Na+ channel 1) and ASIC (acid-sensing ion channel). Biochem J. 361(Pt 3): 443-450
53. Duggan A, Garcia-Anoveros J, Corey DP. The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons. J. Biol. Chem. 2002. 277(7): 5203-5208.
54. Anzai N, Deval E, Schaefer L, et al. The multivalent PDZ domain-containing protein CIPP is a partner of acid-sensing ion channel 3 in sensory neurons. J. Biol. Chem. 2002, 277(19): 16655-16661.
55. Yudin YK, Tamarova ZA, Ostrovskaya OI, Moroz LL, Krishtal OA. RFa-related peptides are algogenic: evidence in vitro and in vivo. Eur J Meurosci. 2004, 20(5):1419-1423.
56. Jasi J, Furukawa H, Gonzales EB, Gouaux E. Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH. Nature. 2007, 449(7160): 316-323.