In vivo light-induced and basal phospholipase C activity in Drosophila photoreceptors measured with genetically targeted phosphatidylinositol 4,5-bisphosphate-sensitive ion channels (Kir2.1)
In vivo light-induced and basal phospholipase C activity in Drosophila photoreceptors measured with genetically targeted phosphatidylinositol 4,5-bisphosphate-sensitive ion channels (Kir2.1)
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DOI：
10.1074/jbc.m407525200
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发表时间：
2004-11-12
影响因子：
4.8
通讯作者：
Raghu, P
中科院分区：
文献类型：
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作者：
Hardie, RC;Gu, YC;Raghu, P
果蝇感光细胞表达磷脂酰肌醇4，5-二磷酸(PIP2)敏感的内向整流通道Kir2.1，并用于体内PIP2水平的监测。由于野生型(WT)Kir2.1通道似乎被当时的PIP2浓度饱和，我们进行了单一氨基酸取代(R228Q)，这降低了对PIP2的有效亲和力，产生的通道产生的电流与与光转导相关的PIP2水平成比例。为了分离Kir2.1电流，记录了缺乏两类光敏瞬时受体电位通道(Trp和TRPL)的突变体。光导致磷脂酶C(PLC)在每个吸收的光子中以大约三到四个微绒毛的速度有效地消耗PIP2，速度超过每秒总微绒毛磷脂的150%。重新合成PIP2的半衰期类似于50 S。当细胞中的三磷酸腺苷被阻断后，KIR电流以最大的速率自发衰减，相当于每分钟损失总PIP2的40%。这主要归因于基础PLC活性的下降，因为在缺乏PLC的Norpa突变体中，PLC活性大大降低。我们试图通过使用PLC抑制剂U73122来证实这一点；然而，我们发现这是一种新的Kir2.1通道抑制剂。二酰甘油激酶突变体(RdgA)的PIP2水平降低了5倍，但基础PLC活性仍然显著，这与二酰甘油水平升高是该突变体的结构性Trp通道活动特征有关的提示一致。
The phosphatidylinositol 4,5-bisphosphate (PIP2)-sensitive inward rectifier channel Kir2.1 was expressed in Drosophila photoreceptors and used to monitor in vivo PIP2 levels. Since the wild-type (WT) Kir2.1 channel appeared to be saturated by the prevailing PIP2 concentration, we made a single amino acid substitution (R228Q), which reduced the effective affinity for PIP2 and yielded channels generating currents proportional to the PIP2 levels relevant for phototransduction. To isolate Kir2.1 currents, recordings were made from mutants lacking both classes of light-sensitive transient receptor potential channels (TRP and TRPL). Light resulted in the effective depletion of PIP2 by phospholipase C (PLC) in approximately three or four microvilli per absorbed photon at rates exceeding similar to150% of total microvillar phosphoinositides per second. PIP2 was resynthesized with a half-time of similar to50 s. When PIP2 resynthesis was prevented by depriving the cell of ATP, the Kir current spontaneously decayed at maximal rates representing a loss of similar to40% loss of total PIP2 per minute. This loss was attributed primarily to basal PLC activity, because it was greatly decreased in norpA mutants lacking PLC. We tried to confirm this by using the PLC inhibitor U73122; however, this was found to act as a novel inhibitor of the Kir2.1 channel. PIP2 levels were reduced similar to5-fold in the diacylglycerol kinase mutant (rdgA), but basal PLC activity was still pronounced, consistent with the suggestion that raised diacylglycerol levels are responsible for the constitutive TRP channel activity characteristic of this mutant.