Antagonistic regulation of native Ca2+- and ATP-sensitive cation channels in brain capillaries by nucleotides and decavanadate.
Antagonistic regulation of native Ca2+- and ATP-sensitive cation channels in brain capillaries by nucleotides and decavanadate.
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核苷酸和decavanate对脑毛细血管中天然Ca2+ - 和ATP敏感阳离子通道的拮抗调节。
DOI：
10.1085/jgp.200309008
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发表时间：
2004-06
影响因子：
3.8
通讯作者：
Adam-Vizi, Vera
中科院分区：
文献类型：
--
作者：
Csanady, Laszlo;Adam-Vizi, Vera
在大鼠脑微血管内皮细胞上，观察胞浆核苷酸对钙敏感和三磷酸腺苷敏感的非选择性阳离子通道(CA-NSCs)的调节作用。胞内核苷酸对三磷酸腺苷、腺苷二磷酸和腺苷三磷酸腺苷的表观Ki值分别为17、9和2μM，这是高亲和力抑制通道开放速率和低亲和力刺激关闭速率的结果。细胞内[Ca~(2+)]和电压影响ATP对Po的抑制，但不影响其开放速率，提示核苷酸结合位点的构象只受通道门状态的影响，而不受Ca~(2+)和电压传感器的影响。ATP抑制作用不受通道衰竭的影响。核苷酸结构影响对碱基取代不敏感的抑制效力，但3‘-5’环化、去除所有磷酸盐或完全省略碱基会大大降低抑制效力。反之，十钒酸能有效地(K1/2=90 nM)刺激Po，并在功能上与抑制性核苷酸竞争。根据动力学分析，我们得出结论：(A)ATP、ADP和AMP与一个共同的位点结合；(B)核苷酸通过简单的可逆结合而发生抑制，这是由于与闭合通道的结合比开放通道构象更紧密；(C)核苷酸结合位点的构象不直接受钙离子和电压的调节；(D)ATP、ADP和AMP抑制效力的差异反映了它们对闭合通道的不同亲和力；和(E)尽管十钒酸是迄今为止发现的唯一一种化合物，即使在存在毫厘核苷酸的情况下，也能以高亲和力刺激Po，显然是通过竞争核苷酸结合位点来实现的，但类似的机制可能允许活细胞中的CA-NSC通道开放，尽管存在生理水平的核苷酸。十钒酸现在为研究天然CA-NSC通道和筛选克隆通道提供了一个有价值的工具。
Regulation by cytosolic nucleotides of Ca2+- and ATP-sensitive nonselective cation channels (CA-NSCs) in rat brain capillary endothelial cells was studied in excised inside-out patches. Open probability (Po) was suppressed by cytosolic nucleotides with apparent KI values of 17, 9, and 2 μM for ATP, ADP, and AMP, as a consequence of high-affinity inhibition of channel opening rate and low-affinity stimulation of closing rate. Cytosolic [Ca2+] and voltage affected inhibition of Po, but not of opening rate, by ATP, suggesting that the conformation of the nucleotide binding site is influenced only by the state of the channel gate, not by that of the Ca2+ and voltage sensors. ATP inhibition was unaltered by channel rundown. Nucleotide structure affected inhibitory potency that was little sensitive to base substitutions, but was greatly diminished by 3′-5′ cyclization, removal of all phosphates, or complete omission of the base. In contrast, decavanadate potently (K1/2 = 90 nM) and robustly stimulated Po, and functionally competed with inhibitory nucleotides. From kinetic analyses we conclude that (a) ATP, ADP, and AMP bind to a common site; (b) inhibition by nucleotides occurs through simple reversible binding, as a consequence of tighter binding to the closed-channel relative to the open-channel conformation; (c) the conformation of the nucleotide binding site is not directly modulated by Ca2+ and voltage; (d) the differences in inhibitory potency of ATP, ADP, and AMP reflect their different affinities for the closed channel; and (e) though decavanadate is the only example found to date of a compound that stimulates Po with high affinity even in the presence of millimolar nucleotides, apparently by competing for the nucleotide binding site, a comparable mechanism might allow CA-NSC channels to open in living cells despite physiological levels of nucleotides. Decavanadate now provides a valuable tool for studying native CA-NSC channels and for screening cloned channels.