Stevens Bruce R, Feldman Daniel H, Liu Zhilin, Harvey William R
The Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, FL 32652, USA.
J Exp Biol. 2002 Aug;205(Pt 16):2545-53. doi: 10.1242/jeb.205.16.2545.
CAATCH1 functions both as an amino-acid-gated cation channel and as a cation-dependent, proline-preferring, nutrient amino acid transporter in which the two functions are thermodynamically uncoupled. This study focuses on the ionic channel aspect, in which a Tyr(147) (wild type) to Phe(147) (Y147F) site-directed mutation was investigated by steady-state electrophysiological measurements in the Xenopus laevis oocyte expression system. This tyrosine residue is conserved within the third transmembrane domain in members of the Na(+):neurotransmitter transporter family (SNF), where it plays a role in binding pharmacological ligands such as cocaine to the serotonin (SERT), dopamine (DAT) and norepinephrine (NET) transporters. Epithelial CAATCH1 is a member of the SNF family. The results show that amino acid ligand-gating selectivity and current magnitudes in Na(+)- and K(+)-containing media are differentially altered in CAATCH1 Y147F compared with the wild type. In the absence of amino acid ligands, the channel conductance of Na(+), K(+) and Li(+) that is observed in the wild type was reduced to virtually zero in Y147F. In the wild type, proline binding increased conductance strongly in Na(+)-containing medium and moderately in K(+)-containing medium, whereas in Y147F proline failed to elicit any cation currents beyond those of N-methyl-D-glucamine- or water-injected oocytes. In the wild type, methionine binding strongly inhibited inward Na(+) currents, whereas in Y147F it strongly stimulated inward currents in both Na(+) and K(+)-containing media. Indeed, in Na(+)-containing medium, the relative potency ranking for inward current inhibition in the wild type (Met>Leu>Gly>Phe>Thr) was similar to the ranking of ligand-permissive gating of large inward currents in Y147F. In Na(+)-containing medium, current/voltage relationships elicited by ligands in the wild type were complex and reversing, whereas in Y147F they were linear and inwardly rectifying. In K(+)-containing medium, current/voltage relationships remained non-linear in Y147F. Both wild-type and Y147F currents were Cl(-)-independent. Together, these data demonstrate a critical role for Tyr(147) in ligand-binding selectivity and modulation of the ionic channel conductance in CAATCH1. The results support the argument that inhibition of the CAATCH1 conductance by free methionine shares some properties in common with ligand inhibition of DAT, SERT, NET and the gamma-aminobutyric acid transporter (GAT1).
CAATCH1既作为氨基酸门控阳离子通道，又作为阳离子依赖性、脯氨酸偏好性的营养氨基酸转运体发挥作用，其中这两种功能在热力学上是不耦合的。本研究聚焦于离子通道方面，通过在非洲爪蟾卵母细胞表达系统中进行稳态电生理测量，研究了酪氨酸（147）（野生型）到苯丙氨酸（147）（Y147F）的定点突变。该酪氨酸残基在Na⁺:神经递质转运体家族（SNF）成员的第三个跨膜结构域中保守，在那里它在诸如可卡因等药理学配体与5-羟色胺（SERT）、多巴胺（DAT）和去甲肾上腺素（NET）转运体的结合中起作用。上皮CAATCH1是SNF家族的成员。结果表明，与野生型相比，CAATCH1 Y147F中氨基酸配体门控选择性和在含Na⁺和K⁺介质中的电流大小有差异地改变。在没有氨基酸配体的情况下，野生型中观察到的Na⁺、K⁺和Li⁺的通道电导在Y147F中几乎降至零。在野生型中，脯氨酸结合在含Na⁺介质中强烈增加电导，在含K⁺介质中适度增加电导，而在Y147F中，脯氨酸除了在注射N-甲基-D-葡糖胺或水的卵母细胞中引发的阳离子电流外，未能引发任何阳离子电流。在野生型中，甲硫氨酸结合强烈抑制内向Na⁺电流，而在Y147F中，它在含Na⁺和K⁺介质中均强烈刺激内向电流。实际上，在含Na⁺介质中，野生型中内向电流抑制的相对效力排名（甲硫氨酸>亮氨酸>甘氨酸>苯丙氨酸>苏氨酸）与Y147F中大量内向电流的配体允许门控排名相似。在含Na⁺介质中，野生型中配体引发的电流/电压关系复杂且可逆，而在Y147F中它们是线性的且内向整流。在含K⁺介质中，Y147F中的电流/电压关系仍然是非线性的。野生型和Y147F电流均与Cl⁻无关。总之，这些数据证明了酪氨酸（147）在CAATCH1的配体结合选择性和离子通道电导调节中起关键作用。结果支持了游离甲硫氨酸对CAATCH1电导的抑制与DAT、SERT、NET和γ-氨基丁酸转运体（GAT1）的配体抑制有一些共同特性的观点。
