Differential regulation of the InsP₃ receptor type-1 and -2 single channel properties by InsP₃, Ca²⁺ and ATP.
Differential regulation of the InsP₃ receptor type-1 and -2 single channel properties by InsP₃, Ca²⁺ and ATP.
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InsPα、Ca2α 和 ATP 对 InsPα 受体 1 型和 -2 型单通道特性的差异调节。
DOI：
10.1113/jphysiol.2012.228320
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发表时间：
2012
期刊：
影响因子：
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通讯作者：
Yule,DavidI
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作者：
Wagner2nd,LarryE;Yule,DavidI
要点·1，4，5-三磷酸肌醇受体（InsP3 Rs）的三个家族成员代表了普遍表达的细胞内Ca 2+释放通道。这些通道的活性由InsP3、Ca2+和ATP以协调和相互关联的方式调节。目前还不确定是否每种同种型是由这些配体以相同的方式调节。在这项研究中，我们直接比较了在这些配体存在下分离表达的哺乳动物InsP3R-1和InsP3R-2的单通道活性。每种亚型的活性增加是由从静止的"停放"状态到以爆发活性为特征的"驱动"模式的转变介导的。配体在这些爆发期间不影响单通道活动，而是调节爆发活动的程度。·动力学分析表明，在InsP3R-1vs中，Ca2+和ATP通过不同的机制调节向爆发活性的转变。InsP3R-2：尽管这两个通道的活性都受到Ca 2+的双相调节，并且[InsP3]的变化并没有改变这种关系，但ATP升高会增加InsP3 R-1活性的Ca 2+敏感性，而不会增加通道的最大可实现开放概率（Po）。相比之下，ATP只是增加了InsP3R-2的最大可吸收功率，而不改变其对Ca2+的敏感性。InsP3R-1和InsP3R-2的不同调控模式可能会显着影响细胞内Ca 2+信号的特征，在这些亚型中观察到的cells expressed.AbstractAn作为InsP3受体（InsP3R）活性的结果，细胞内Ca 2+水平的升高代表了一个普遍存在的信号通路控制各种各样的细胞事件。InsP3R活性受主要配体InsP3、Ca2+和ATP水平的严格控制。重要的是，InsP3R以双相方式调节。通过所有InsP3R家族成员的Ca2+释放也受到ATP的显著调节，尽管具有亚型特异性。为了确定是否有一种共同的机制可以解释这些InsP3R家族成员的ATP和Ca2+调节，我们检查了[ATP]对DT40 - 3KO细胞中表达的大鼠InsP3R-1（rInsP3R-1）和小鼠InsP3R-2（mInsP3R-2）活性的Ca2+依赖性的影响。我们在膜片钳中使用不同的[ATP]，[InsP3]和[Ca2 +]的核上膜片钳记录技术，并在稳定转染的DT 40细胞中测量单个InsP3R通道活性。在相同条件下，在饱和[InsP3]和[ATP]时，rInsP3R-1和mInsP3R-2的活性在单通道电导、最大可达开放概率（Po）和激活和抑制活性所需的[Ca2 +]方面基本相同。然而，与[InsP3]饱和时的rInsP3R-1相反，mInsP3R-2的活性不受[ATP]的影响。在[InsP3]较低时，ATP对mInsP3R-2Po有显著影响，但与rInsP3R-1不同，这不是通过改变相对Ca2+依赖性而发生的，而是通过简单地增加特定[InsP3]和[Ca2 +]的最大可吸收Poat。[InsP3]未改变rInsP3R-1或mInsP3R-2中Ca2+对活性的双相调节。单通道动力学分析表明，Ca~（2+）和ATP主要通过促进通道的延长爆发活性来调节Pop-predominately，但每个受体的潜在生物物理机制似乎不同。InsP3R通道活性的亚型特异性调节可能有助于表达这些受体亚型的细胞中Ca2+信号传导的保真度。
Key points•Three family members of inositol 1,4,5‐trisphosphate receptors (InsP3Rs) represent ubiquitously expressed intracellular Ca2+release channels. The activity of the channels is regulated in a concerted and inter‐related fashion by InsP3, Ca2+and ATP. It is not established whether each isoform is regulated by these ligands in an identical fashion. In this study we directly compare the single channel activity of mammalian InsP3R‐1 and InsP3R‐2 expressed in isolation in the presence of these ligands.•An increase in activity for each isoform was mediated by a transition from a quiescent, ‘parked’ state to a ‘drive’ mode characterized by bursting activity. Ligands did not affect the single channel activity during these bursts but instead modulate the extent of bursting activity.•Kinetic analysis revealed that the regulation of the transition to bursting activity by Ca2+and ATP occurred by different mechanisms in InsP3R‐1vs. InsP3R‐2: although the activity of both channels was biphasically regulated by Ca2+and changes in [InsP3] did not alter this relationship, elevated ATP increased the Ca2+sensitivity of InsP3R‐1 activity without increasing the maximal achievable open probability (Po) of the channel. In contrast, ATP simply increased the maximal achievablePowithout altering Ca2+sensitivity of InsP3R‐2.•The differing modes of regulation of InsP3R‐1 and InsP3R‐2 probably markedly influence the characteristics of intracellular Ca2+signals observed in cells in which these isoforms are expressed.AbstractAn elevation of intracellular Ca2+levels as a result of InsP3receptor (InsP3R) activity represents a ubiquitous signalling pathway controlling a wide variety of cellular events. InsP3R activity is tightly controlled by the levels of the primary ligands, InsP3, Ca2+and ATP. Importantly, InsP3Rs are regulated by in a biphasic manner. Ca2+release through all InsP3R family members is also modulated dramatically by ATP, albeit with sub‐type‐specific properties. To ascertain if a common mechanism can account for ATP and Ca2+regulation of these InsP3R family members, we examined the effects of [ATP] on the Ca2+dependency of rat InsP3R‐1 (rInsP3R‐1) and mouse InsP3R‐2 (mInsP3R‐2) activity expressed in DT40‐3KO cells. We used the on‐nucleus patch clamp recording technique with various [ATP], [InsP3] and [Ca2+] in the patch pipette and measured single InsP3R channel activity in stably transfected DT40 cells. Under identical conditions, at saturating [InsP3] and [ATP], the activity of rInsP3R‐1 and mInsP3R‐2 was essentially identical in terms of single channel conductance, maximal achievable open probability (Po) and the [Ca2+] required for activation and inhibition of activity. However, in contrast to rInsP3R‐1 at saturating [InsP3], the activity of mInsP3R‐2 was unaffected by [ATP]. At lower [InsP3], ATP had dramatic effects on mInsP3R‐2Po, but unlike the rInsP3R‐1, this did not occur by altering the relative Ca2+dependency, but by simply increasing the maximally achievablePoat a particular [InsP3] and [Ca2+]. [InsP3] did not alter the biphasic regulation of activity by Ca2+in either rInsP3R‐1 or mInsP3R‐2. Analysis of the single channel kinetics indicated that Ca2+and ATP modulate thePopredominately by facilitating extended bursting activity of the channel but the underlying biophysical mechanism appears to be distinct for each receptor. Subtype‐specific regulation of InsP3R channel activity probably contributes to the fidelity of Ca2+signalling in cells expressing these receptor subtypes.