Functional roles of charged amino acid residues on the wall of the cytoplasmic pore of Kir2.1
Functional roles of charged amino acid residues on the wall of the cytoplasmic pore of Kir2.1
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DOI：
10.1085/jgp.200509434
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发表时间：
2006-04-01
影响因子：
3.8
通讯作者：
Kubo, Y
中科院分区：
文献类型：
--
作者：
Fujiwara, Y;Kubo, Y
已知通过内向整流性K+通道（Kir）的电流整流主要是由于细胞质Mg 2+和多胺对外向电流的阻断。Kir2.1胞质区晶体结构的分析显示，两者的存在都是负的。（E224、D255、D259和E299）和阳性（R228和R260）Kir2.1的细胞质孔壁上的带电残基，但关于这些带电残基的贡献的细节尚不清楚，特别是正电荷，到内部的调整。因此，我们使用单/双点突变体分析了这些带电氨基酸的功能意义，以便更好地理解Kir2.1电流内向整流的结构基础机制。作为第一步，我们使用双电极电压钳检查内向整流系统制备的突变体，其中一个或两个带负电荷或正电荷的氨基酸被取代中和。我们发现，内向整流的强度往往是由细胞质孔内的净负电荷。然后，我们使用由内而外切除膜片钳记录，分析细胞内阻滞剂和K+渗透的阻断突变的效果。我们观察到，细胞质孔内的净负电荷的减少降低了通道的敏感性，封锁由Mg 2+或精胺和封锁的电压依赖性。它还减少了K+渗透;即，在完全不存在胞质阻滞剂的情况下，它降低了单通道电导，增加了开放通道噪声，并加强了内在内向整流。综上所述，这些数据表明，Kir的带负电荷的细胞质孔静电地聚集阳离子，如Mg 2+、精胺和K+，使得跨膜孔被K+离子充分填充，这使得能够具有足够的外向K+电导的强电压依赖性阻断。
It is known that rectification of currents through the inward rectifier K+ channel (Kir) is mainly due to blockade of the outward current by cytoplasmic Mg2+ and polyamines. Analyses of the crystal structure of the cytoplasmic region of Kir2.1 have revealed the presence of both negatively (E224, D255, D259, and E299) and positively (R228 and R260) charged residues on the wall of the cytoplasmic pore of Kir2.1, but the detail is not known about the contribution of these charged residues, the positive charges in particular, to the inward rectification. We therefore analyzed the functional significance of these charged amino acids using single/double point mutants in order to better understand the structure-based mechanism underlying inward rectification of Kir2.1 currents. As a first step, we used two-electrode voltage clamp to examine inward rectification in systematically prepared mutants in which one or two negatively or positively charged amino acids were neutralized by substitution. We found that the intensity of the inward rectification tended to be determined by the net negative charge within the cytoplasmic pore. We then used inside-out excised patch clamp recording to analyze the effect of the mutations on blockade by intracellular blockers and on K+ permeation. We observed that a decrease in the net negative charge within the cytoplasmic pore reduced both the susceptibility of the channel to blockade by Mg2+ or spermine and the voltage dependence of the blockade. It also reduced K+ permeation; i.e., it decreased single channel conductance, increased open-channel noise, and strengthened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these data suggest that the negatively charged cytoplasmic pore of Kir electrostatically gathers cations such as Mg2+, spermine, and K+ so that the transmembrane pore is sufficiently filled with K+ ions, which enables strong voltage-dependent blockade with adequate outward K+ conductance.