Rebrik Tatiana I, Korenbrot Juan I
Department of Physiology, School of Medicine, University of California at San Francisco, San Francisco, CA 94143, USA.
J Gen Physiol. 2004 Jan;123(1):63-75. doi: 10.1085/jgp.200308952.
In the mammalian retina, cone photoreceptors efficiently adapt to changing background light intensity and, therefore, are able to signal small differences in luminance between objects and backgrounds, even when the absolute intensity of the background changes over five to six orders of magnitude. Mammalian rod photoreceptors, in contrast, adapt very little and only at intensities that nearly saturate the amplitude of their photoresponse. In search of a molecular explanation for this observation we assessed Ca2+-dependent modulation of ligand sensitivity in cyclic GMP-gated (CNG) ion channels of intact mammalian rods and cones. Solitary photoreceptors were isolated by gentle proteolysis of ground squirrel retina. Rods and cones were distinguished by whether or not their outer segments bind PNA lectin. We measured membrane currents under voltage-clamp in photoreceptors loaded with Diazo-2, a caged Ca2+ chelator, and fixed concentrations of 8Br-cGMP. At 600 nM free cytoplasmic Ca2+ the midpoint of the cone CNG channels sensitivity to 8BrcGMP, 8BrcGMPK1/2, is approximately 2.3 microM. The ligand sensitivity is less in rod than in cone channels. Instantly decreasing cytoplasmic Ca2+ to <30 nM activates a large inward membrane current in cones, but not in rods. Current activation arises from a Ca2+ -dependent modulation of cone CNG channels, presumably because of an increase in their affinity to the cyclic nucleotide. The time course of current activation is temperature dependent; it is well described by a single exponential process of approximately 480 ms time constant at 20-21 degrees C and 138 ms at 32 degrees C. The absence of detectable Ca2+-dependent CNG current modulation in intact rods, in view of the known channel modulation by calmodulin in-vitro, affirms the modulation in intact rods may only occur at low Ca2+ concentrations, those expected at intensities that nearly saturate the rod photoresponse. The correspondence between Ca2+ dependence of CNG modulation and the ability to light adapt suggest these events are correlated in photoreceptors.
在哺乳动物视网膜中，视锥光感受器能有效地适应不断变化的背景光强度，因此，即使背景光的绝对强度变化了五到六个数量级，它们也能够感知物体与背景之间亮度的微小差异。相比之下，哺乳动物的视杆光感受器适应能力很弱，且仅在几乎使光反应幅度饱和的强度下才会产生适应。为了寻找这一现象的分子学解释，我们评估了完整的哺乳动物视杆和视锥细胞中环鸟苷酸门控（CNG）离子通道中配体敏感性的钙依赖性调节。通过对花栗鼠视网膜进行温和的蛋白酶解来分离单个光感受器。视杆和视锥细胞通过其外段是否结合花生凝集素（PNA）来区分。我们在电压钳制下测量了装载有重氮-2（一种笼形钙螯合剂）和固定浓度8-溴环鸟苷酸（8Br-cGMP）的光感受器的膜电流。在游离细胞质钙浓度为600 nM时，视锥细胞CNG通道对8-溴环鸟苷酸的敏感性中点（8Br-cGMP K1/2）约为2.3 μM。视杆细胞通道中的配体敏感性低于视锥细胞通道。将细胞质钙瞬间降低至<30 nM会在视锥细胞中激活一个大的内向膜电流，但在视杆细胞中不会。电流激活源于视锥细胞CNG通道的钙依赖性调节，可能是因为其对环核苷酸的亲和力增加。电流激活的时间进程取决于温度；在20 - 21℃时，它可以用一个时间常数约为480 ms的单指数过程很好地描述，在32℃时为138 ms。鉴于已知钙调蛋白在体外对通道有调节作用，而完整视杆细胞中未检测到钙依赖性CNG电流调节，这表明完整视杆细胞中的调节可能仅在低钙浓度下发生，即在几乎使视杆光反应饱和的强度下预期的钙浓度。CNG调节的钙依赖性与光适应能力之间的对应关系表明，这些事件在光感受器中是相关的。
