Intrinsic electrostatic potential in the BK channel pore: Role in determining single channel conductance and block
Intrinsic electrostatic potential in the BK channel pore: Role in determining single channel conductance and block
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DOI：
10.1085/jgp.200709862
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发表时间：
2008-02-01
影响因子：
3.8
通讯作者：
Latorre, Ramon
中科院分区：
文献类型：
--
作者：
Carvacho, Ingrid;Gonzalez, Wendy;Latorre, Ramon
大电导Ca 2+电压激活的K+（BK）通道的内前庭含有在较低电导的K+通道（hSlo中的Glu 386和Glu 389）中不存在的8个负电荷的环，其通过静电机制调节通道电导（Brelidze，T.一、X. Niu和K. L.麦格尔比2003. Proc. Natl. Acad. Sci. USA. 100：9017-9022）。在BK通道中，在细胞外环中也存在两个酸性氨基酸残基（hSlo中的Asp 326和Glu 329）。为了确定这些电荷对通道电导的静电影响，我们在非洲爪蟾卵母细胞上表达野生型BK通道和突变体E386 N/E389 N、D326 N、E329 Q和D326 N/E329 Q通道，并在膜片钳下测量表达的电流。E329对电导的贡献可以忽略不计，在对称的110 mM K+中在0 mV下测量的D326 N/E329 Q通道的单通道电导比对照低18%。电流-电压曲线显示D326 N和双突变体的弱外向整流。突变体和野生型BK之间的电导差异是由静电效应引起的，因为它们在低K+（30 mM）时增强，在高K+（1 M K+）时消失。我们确定静电势的变化，Δ phi，所造成的电荷中和使用TEA（+）块的细胞外电荷和Ba 2+的细胞内电荷。当关闭细胞外电荷时，我们测量到TEA（+）位点处的Delta phi为13 +/- 2 mV，当关闭细胞内电荷时，Ba 2+位点处的Delta phi为17 +/- 2 mV。为了理解电荷中和的静电效应，我们使用嵌入脂质双层中的BK通道分子模型并求解泊松-玻尔兹曼方程来确定Δ phi。该模型充分解释了实验结果，特别是，给出了一个经济的解释的差分效应的电导的电荷D326和E329的中和。
The internal vestibule of large-conductance Ca2+ voltage-activated K+ (BK) channels contains a ring of eight negative charges not present in K+ channels of lower conductance (Glu386 and Glu389 in hSlo) that modulates channel conductance through an electrostatic mechanism (Brelidze, T. I., X. Niu, and K. L. Magleby. 2003. Proc. Natl. Acad. Sci. USA. 100: 9017-9022). In BK channels there are also two acidic amino acid residues in an extracellular loop (Asp326 and Glu329 in hSlo). To determine the electrostatic influence of these charges on channel conductance, we expressed wild-type BK channels and mutants E386N/E389N, D326N, E329Q, and D326N/E329Q channels on Xenopus laevis oocytes, and measured the expressed currents under patch clamp. Contribution of E329 to the conductance is negligible and single channel conductance of D326N/E329Q channels measured at 0 mV in symmetrical 110 mM K+ was 18% lower than the control. Current-voltage curves displayed weak outward rectification for D326N and the double mutant. The conductance differences between the mutants and wild-type BK were caused by an electrostatic effect since they were enhanced at low K+ (30 mM) and vanished at high K+ (1 M K+). We determine the electrostatic potential change, Delta phi, caused by the charge neutralization using TEA(+) block for the extracellular charges and Ba2+ for intracellular charges. We measured 13 +/- 2 mV for Delta phi at the TEA(+) site when turning off the extracellular charges, and 17 +/- 2 mV for the Delta phi at the Ba2+ site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined Delta phi using a BK channel molecular model embedded in a lipid bilayer and solving the Poisson-Boltzmann equation. The model explains the experimental results adequately and, in particular, gives an economical explanation to the differential effect on the conductance of the neutralization of charges D326 and E329.