Li Zhiyuan, Migita Keisuke, Samways Damien S K, Voigt Mark M, Egan Terrance M
Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA.
J Neurosci. 2004 Aug 18;24(33):7378-86. doi: 10.1523/JNEUROSCI.1423-04.2004.
ATP opens ionotropic P2X channels through a process that is poorly understood. We made an array of mutant rat P2X2 channels containing unique alanine substitutions in the transmembrane segments with the goal of identifying possible secondary structure and mapping gating domains in the pore. Alteration of channel function was measured as a change in ATP potency, 2'-3'-O-(4-benzoylbenzoyl)ATP (BzATP) efficacy, and deactivation kinetics. Four mutants (V45A, Y47A, V51A, and D349A) failed to respond to ATP. Seven (H33A, Q37A, I40A, L41A, Y43A, F44A, and I50A) of 22 mutations in the first transmembrane segment (TM1) produced channels with altered potencies, and two mutants (Y43A and F44A) were active in the absence of agonist. The pattern of hits was consistent with a helical secondary structure. In contrast, nine (I328A, P329A, N333A, L338A, T339A, S340A, G342A, G344A, and S345A) of 24 mutations in the second transmembrane segment (TM2) resulted in a change in potency, but no regular pattern of impact was apparent. Many of the same mutations that altered ATP potency also changed the relative efficacy of the partial agonist BzATP. Together, these data suggest that both TM1 and TM2 participate in the conformational changes that occur during receptor activation and help to define domains involved in conformational switching within or near the pore.
三磷酸腺苷(ATP)通过一个尚不清楚的过程开启离子型P2X通道。我们制作了一系列突变大鼠P2X2通道,这些通道在跨膜区段含有独特的丙氨酸替代,目的是识别可能的二级结构并绘制孔道中的门控结构域。通道功能的改变通过ATP效力、2'-3'-O-(4-苯甲酰苯甲酰)ATP(BzATP)效力和失活动力学的变化来衡量。四个突变体(V45A、Y47A、V51A和D349A)对ATP无反应。在第一个跨膜区段(TM1)的22个突变中有7个(H33A、Q37A、I40A、L41A、Y43A、F44A和I50A)产生了效力改变的通道,并且两个突变体(Y43A和F44A)在没有激动剂的情况下具有活性。命中模式与螺旋二级结构一致。相比之下,在第二个跨膜区段(TM2)的24个突变中有9个(I328A、P329A、N333A、L338A、T339A、S340A、G342A、G344A和S345A)导致效力改变,但没有明显的规律影响模式。许多改变ATP效力的相同突变也改变了部分激动剂BzATP的相对效力。总之,这些数据表明TM1和TM2都参与受体激活过程中发生的构象变化,并有助于确定孔道内或其附近参与构象转换的结构域。