Two functionally distinct forms of guanosine cyclic 3',5'-phosphate stimulated cation channels in a bovine rod photoreceptor disk preparation.
Two functionally distinct forms of guanosine cyclic 3',5'-phosphate stimulated cation channels in a bovine rod photoreceptor disk preparation.
复制标题
两种功能不同形式的环状鸟苷 3,5-磷酸刺激牛视杆感光盘制剂中的阳离子通道。
DOI：
10.1021/bi00412a029
复制
发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Goldin,SM
中科院分区：
文献类型：
--
作者：
Pearce,LB;Calhoon,RD;Burns,PR;Vincent,A;Goldin,SM
生物化学和分子药理学系，哈佛医学院，波士顿，马萨诸塞州02115接收于1987年9月4日;修订的Mandarin pt接收于1988年2月18日摘要：通过一种新的快速灌流方法，在25-100-ms的时间尺度上测量了环核苷酸刺激的22 Na+和45 Ca 2+从纯化的牛棒外节盘制备物中的流出。通过8-溴鸟苷环S "-磷酸（8-Br-cGMP）的阳离子外排的激活在25 ms内是最大的。在宽范围的8-Br-cGMP浓度内，外排终止的动力学精确地符合两个指数衰减过程的总和：快速阶段（衰减常数为200 ms）和较慢阶段（衰减常数为1.6 s）。流出的双相衰减的动力学不能被解释为可释放的22 Na池的耗尽，但似乎反映了通道失活的内在过程。8-Br-cGMP刺激释放的积极积累的45 Ca表现出相同的双相衰减动力学。Ca释放的最大速率[5 nmol-（mg盘蛋白）-1-min_1]可能足以在20 ms内产生局部细胞质[Ca]的1 pM变化。两个衰变阶段的Ca：Na选择性比为-0.5：1。8-Br-cGMP显示出较低的效力（EC 50为8.4 μ M对2.8 μ M），但在其激活22 Na流出的快速衰减相对缓慢衰减相方面具有较高程度的协同性。衰减的较慢阶段被25 μ M/-顺式-地尔硫卓选择性地抑制，这是快速衰减阶段的相对较弱的抑制剂。钠离子（5-10 mM）选择性地抑制8-Br-cGMP刺激的~（45）Ca释放的快速衰减相。假设这两个动力学上不同的衰变阶段代表了cGMP刺激的阳离子通道的两种功能上不同的形式。脊椎动物视杆细胞外节（ROS）1的盘膜中视紫红质的耳溶解引发一系列事件，导致ROS质膜的超极化[Stryer（1986）综述]。最近在ROS质膜上发现cGMP激活的离子通道（Fesenko，1985; Yau & Nakatani，1985），强烈支持这样的假设，即细胞质游离cGMP水平的降低通过光诱导的环核苷酸磷酸二酯酶的激活产生这种快速的超极化。这是基于对冷冻和超声处理的ROS膜囊泡中cGMP刺激的Ca 2+摄取的观察（Caretta & Cavagionni，1983），cGMP诱导的Ca 2+从细胞裂解或渗透的ROS中释放（Koch & Kaupp，1985），以及cGMP刺激的Ca 2+从纯化的光感受器盘制备物中释放（Puckett & Goldin，1986）。在后一项研究中，Ca 2+从由单独的ATP依赖性Ca 2+摄取活性主动积累的Ca 2+库中释放（Puckett等人，1985年）。ROS中的细胞质游离[Ca 2 +]是亚微摩尔的（McNaughton等人，1986;米勒& Korenbrot，1986），而圆盘含有高水平的Ca 2+，超过2 mM（Kaupp & Schnetkamp，1982; Schroder & Fain，1984）。在此基础上，可以预期，
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 Received September 4, 1987; Revised Manuscript Received February 18, 1988 abstract: Cyclic nucleotide stimulated efflux of 22Na+ and 45Ca2+ from a purified bovine rod outer segment disk preparation was measured on the 25-100-ms time scale by a novel rapid superfusion method. Activation of cation efflux by 8-bromoguanosine cyclic S'^'-phosphate (8-Br-cGMP) was maximal within 25 ms. Over a wide range of concentrations of 8-Br-cGMP, the kinetics of termination of efflux precisely conformed to the sum of two exponential decay processes: a rapid phase (decay constant of 200 ms) and a slower phase (decay constant of 1.6 s). The kinetics of the biphasic decay of efflux cannot be explained by depletion of a pool of releaseable 22Na but appear to reflect an intrinsic process for inactivation of the channels. 8-Br-cGMP-stimulated release of actively accumulated 45Ca exhibited identical biphasic decay kinetics. The maximum rate of Ca release [5 nmol-(mg of disk protein)-1-min_1] may be sufficient to produce a 1 pM change in local cytoplasmic [Ca] within 20 ms. The Ca: Na selectivity ratio is—0.5: 1 for both decay phases. 8-Br-cGMP demonstrated a lower potency (EC50 of 8.4/uM vs 2.8 jiM) but a higher degree of cooperativity in its activation of the rapid vs the slower decay phase of 22Na efflux. The slower phase of decay was selectively inhibited by 25/iM/-cw-diltiazem, a relatively weak inhibitor of the rapid decay phase. Sodium ion (5-10 mM) selectively inhibited the rapid decay phase of 8-Br-cGMP-stimulated 45Ca release. These two kinetically and pharmacologically distinct phases of decay are hypothesized to represent two functionally distinct forms of cGMP-stimulated cation channels.^^ otolysis of rhodopsin in the disk membrane of vertebrate rod photoreceptor outer segments (ROS) 1 initiates a sequence of events leading to the hyperpolarization of the ROS plasma membrane [reviewed by Stryer (1986)]. The recent discovery and characterization of cGMP-activatedion channels in the ROS plasma membrane (Fesenko, 1985; Yau & Nakatani, 1985) strongly favor the hypothesis that the reduction of free cytoplasmic cGMP levels producesthis rapid hyperpolarization via a light-induced activation of a cyclic nucleotide phosphodiesterase.Photoreceptor disks are also hypothesized to contain cGMP-sensitive channels. This is based on observations of cGMP-stimulated Ca2+ uptake in frozen and sonicated ROS membrane vesicles (Caretta & Cavagionni, 1983), cGMP-induced release of Ca2+ from osmotically lysed or permea-bilized ROS (Koch & Kaupp, 1985), and cGMP-stimulated release of Ca2+ from a purified photoreceptor disk preparation (Puckett & Goldin, 1986). In the latter study, Ca2+ was released from a Ca2+ pool actively accumulated by a separate ATP-dependent Ca2+ uptake activity (Puckett et al., 1985). Cytoplasmic free [Ca2+] inROS is submicromolar (McNaughton et al., 1986; Miller & Korenbrot, 1986), and disks contain high levels of Ca2+, in excess of 2 mM (Kaupp & Schnetkamp, 1982; Schroder & Fain, 1984). On the basis of this and the above, it would be expected that the light-in-