Marchesi Arin, Arcangeletti Manuel, Mazzolini Monica, Torre Vincent
Neurobiology Sector, International School for Advanced Studies (SISSA), Trieste, Italy.
J Physiol. 2015 Feb 15;593(4):857-70. doi: 10.1113/jphysiol.2014.284216. Epub 2015 Jan 7.
Desensitization and inactivation provide a form of short-term memory controlling the firing patterns of excitable cells and adaptation in sensory systems. Unlike many of their cousin K(+) channels, cyclic nucleotide-gated (CNG) channels are thought not to desensitize or inactivate. Here we report that CNG channels do inactivate and that inactivation is controlled by extracellular protons. Titration of a glutamate residue within the selectivity filter destabilizes the pore architecture, which collapses towards a non-conductive, inactivated state in a process reminiscent of the usual C-type inactivation observed in many K(+) channels. These results indicate that inactivation in CNG channels represents a regulatory mechanism that has been neglected thus far, with possible implications in several physiological processes ranging from signal transduction to growth cone navigation.
Ion channels control ionic fluxes across biological membranes by residing in any of three functionally distinct states: deactivated (closed), activated (open) or inactivated (closed). Unlike many of their cousin K(+) channels, cyclic nucleotide-gated (CNG) channels do not desensitize or inactivate. Using patch recording techniques, we show that when extracellular pH (pHo ) is decreased from 7.4 to 6 or lower, wild-type CNGA1 channels inactivate in a voltage-dependent manner. pHo titration experiments show that at pHo < 7 the I-V relationships are outwardly rectifying and that inactivation is coupled to current rectification. Single-channel recordings indicate that a fast mechanism of proton blockage underlines current rectification while inactivation arises from conformational changes downstream from protonation. Furthermore, mutagenesis and ionic substitution experiments highlight the role of the selectivity filter in current decline, suggesting analogies with the C-type inactivation observed in K(+) channels. Analysis with Markovian models indicates that the non-independent binding of two protons within the transmembrane electrical field explains both the voltage-dependent blockage and the inactivation. Low pH, by inhibiting the CNGA1 channels in a state-dependent manner, may represent an unrecognized endogenous signal regulating CNG physiological functions in diverse tissues.
脱敏和失活提供了一种短期记忆形式，可控制可兴奋细胞的放电模式以及感觉系统中的适应性。与许多同类的钾离子通道不同，环核苷酸门控（CNG）通道被认为不会脱敏或失活。在此我们报告，CNG通道确实会失活，且失活受细胞外质子控制。对选择性过滤器内谷氨酸残基的滴定会破坏孔结构的稳定性，该结构会朝着非传导性的失活状态坍塌，此过程类似于在许多钾离子通道中观察到的常见C型失活。这些结果表明，CNG通道中的失活代表了一种迄今被忽视的调节机制，可能在从信号转导到生长锥导航等多个生理过程中具有重要意义。
离子通道通过处于三种功能不同的状态之一来控制跨生物膜的离子通量：去激活（关闭）、激活（开放）或失活（关闭）。与许多同类的钾离子通道不同，环核苷酸门控（CNG）通道不会脱敏或失活。使用膜片钳记录技术，我们发现当细胞外pH（pHo）从7.4降至6或更低时，野生型CNGA1通道会以电压依赖性方式失活。pHo滴定实验表明，在pHo < 7时，电流-电压关系呈外向整流，且失活与电流整流相关。单通道记录表明，质子阻断的快速机制是电流整流的基础，而失活则源于质子化下游的构象变化。此外，诱变和离子替代实验突出了选择性过滤器在电流下降中的作用，表明与在钾离子通道中观察到的C型失活类似。马尔可夫模型分析表明，跨膜电场中两个质子的非独立结合解释了电压依赖性阻断和失活。低pH以状态依赖性方式抑制CNGA1通道，可能代表一种未被认识的内源性信号，可调节不同组织中CNG的生理功能。
