Gavazzo P, Picco C, Eismann E, Kaupp U B, Menini A
Istituto di Cibernetica e Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy.
J Gen Physiol. 2000 Sep;116(3):311-26. doi: 10.1085/jgp.116.3.311.
Upon stimulation by odorants, Ca(2+) and Na(+) enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide-gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the alpha subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide-gated channels play a similar role. We studied the corresponding glutamate (E340) of the alpha subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca(2+) and Mg(2+). E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca(2+) and Mg(2+) powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca(2+) relative to Na(+) was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca(2+) and Mg(2+), and affects channel gating by cAMP more than by cGMP.
在气味剂的刺激下，Ca(2+)和Na(+)通过由cAMP直接门控的通道进入嗅觉感觉神经元的纤毛。环核苷酸门控通道已在多种细胞中被发现，并在过去几年中得到了广泛研究。牛视网膜视杆细胞通道α亚基第363位的谷氨酸残基先前已被证明构成一个对外部二价阳离子阻断很重要的阳离子结合位点，并控制单通道特性。因此，人们推测但未证实，其他环核苷酸门控通道相应位置的谷氨酸残基也发挥类似作用。我们研究了牛嗅觉通道α亚基的相应谷氨酸（E340），以确定其在通道门控、Ca(2+)和Mg(2+)的通透与阻断中的作用。将E340突变为天冬氨酸、甘氨酸、谷氨酰胺或天冬酰胺残基，并在非洲爪蟾卵母细胞中表达的突变通道特性在切除的膜片中进行测量。通过单通道记录，我们证明突变使cGMP或cAMP存在时的开放概率降低，cAMP门控时降低幅度更大。此外，我们观察到突变体E340N呈现两种电导水平。我们发现外部Ca(2+)和Mg(2+)都能强烈阻断野生型和E340D突变体中的电流，而当谷氨酸电荷被中和时，它们 的阻断效力会大幅降低。与野生型通道相比，E340G突变体中由外部Ca(2+)相对于Na(+)携带的内向电流更大。总之，我们已经证实牛嗅觉CNG通道E340位的残基位于孔区域，控制外部Ca(2+)和Mg(2+)的通透与阻断，并且对cAMP门控的影响大于对cGMP门控的影响。
