Dissecting movement of the transmembrane segments of non-gastric proton pump mutants with voltage-clamp fluorometry
Dissecting movement of the transmembrane segments of non-gastric proton pump mutants with voltage-clamp fluorometry
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用电压钳荧光测定法解剖非胃质子泵突变体跨膜片段的运动
DOI：
10.1016/j.bpj.2022.11.2808
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发表时间：
2023
影响因子：
3.4
通讯作者：
Artigas, Pablo
中科院分区：
文献类型：
--
作者：
Self, Daniel;Young, Victoria C.;Nakanishi, Hanayo;Abe, Kazuhiro;Artigas, Pablo
Na ~+，K ~+-ATP酶（NKA）在动物细胞质膜上输出3个Na ~+，输入2个K ~+。非胃H+，K+-ATP酶（ngHKA）在几种上皮细胞的顶膜中输出一个H+和输入一个K+。两种P型泵具有约65%的同一性和几乎相同的催化循环，其由不同离子与两种主要构象（E1，对Na+或H+的高亲和力和E2，对K+的更高亲和力）的结合驱动。最近，我们报道了野生型（WT）ngHKA及其产电突变体K794 A和NKA样K794 S/A797 P/W 940/R949 C（SPWC）突变体在K+闭塞E2状态下的结构。我们还报道了SPWC的AMPCP结合的E1 Cryo-EM结构，这与E1（3 Na+）NKA的结构几乎相同。我们获得了WT（Na+和AlF-ADP）和K794 A（Na+和AMPPCP）结构。虽然WT结构与E2 P状态相同，但AMPPCP结合的K794 A结构与（推测）2 Na+结合具有混合构象。跨膜片段TM 4的P和N结构域以及胞质部分呈E1样构象，而跨膜片段TM 4的A结构域、TM 1-TM 3和管腔部分呈E2样构象。为了评估所有三种结构中移动TM片段的位移，我们在TM 1-TM 2，TM 3 -4或TM 5 -6之间的环中引入了单个Cys残基，用四甲基罗丹明马来酰亚胺（TMRM）标记它们，并在爪蟾卵母细胞中电压钳荧光测定法下评估电流和荧光变化。与WT中的稳定E2状态一致，在TM 1-TM 2中引入的TMRM显示出最小的荧光电压依赖性变化，而用K794 A和SPWC突变体观察到的TM 1-TM 2荧光信号的电压依赖性逐渐接近NKA中观察到的信号。在TM 3 -4和TM 5 -6位置引入TMRM的研究正在进行中。由NSF MCB-2003251资助。
The Na+, K+-ATPase (NKA) extrudes 3 Na+ and imports 2 K+ across the plasmalemma of every animal cell. The non-gastric H+, K+-ATPase (ngHKA) exports one H+ and imports one K+ in the apical membrane of several epithelia. Both P-type pumps have∼ 65% identity and nearly identical catalytic cycles driven by binding of different ions to two major conformations (E1, high affinity for Na+ or H+ and E2 higher affinity for K+). Recently, we reported the structures of the wild type (WT) ngHKA and its electrogenic mutant K794A and the NKA-like K794S/A797P/W940/R949C (SPWC) mutant, in the K+-occluded E2 state. We also reported SPWC's AMPCP-bound E1 Cryo-EM structure, which is nearly identical to that of the E1 (3Na+) NKA. We obtained the WT (Na+ and AlF-ADP) and K794A (Na+ and AMPPCP) structures. While the WT structure was identical to E2P state, the AMPPCP-bound K794A structure with (presumably) 2 Na+ bound has a mixed conformation. The P and N domains as well as cytoplasmic portion of transmembrane segment TM4 shows E1-like conformation, but the A domain, TM1-TM3 and the luminal portion of TM4 took an E2-like conformation. To evaluate displacements of the moving TM segments in all three constructs, we introduced a single Cys residue in the loops between TM1-TM2, TM3-4, or TM5-6, labeled them with tetramethylrhodamine maleimide (TMRM) and evaluated currents and fluorescence changes under voltage clamp fluorometry in Xenopus oocytes. Consistent with the stable E2 state in WT, a TMRM introduced in TM1-TM2 showed minimal voltage dependent changes in fluorescence, while the voltage dependence of the TM1-TM2 fluorescent signals observed with K794A and SPWC mutants were progressively closer to signals observed in NKA. Studies with TMRM introduced at TM3-4 and TM5-6 positions are underway. Funded by NSF MCB-2003251.