Garg Priyanka, Sanguinetti Michael C
Nora Eccles Harrison Cardiovascular Research & Training Institute, University of Utah, Salt Lake City, Utah, USA Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah, USA.
Nora Eccles Harrison Cardiovascular Research & Training Institute, University of Utah, Salt Lake City, Utah, USA Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah, USA Department of Internal Medicine, Division of Cardiovascular Medicine, University of Utah, Salt Lake City, Utah, USA.
Physiol Rep. 2014 Sep 11;2(9). doi: 10.14814/phy2.12118. Print 2014 Sep 1.
Under normal physiological conditions, the open probability of Slo2.1 K(+) channels is low. Elevation of cytosolic [Na(+)] and [Cl(-)] caused by ischemia or rapid electrical pacing of cells increases the open probability of Slo2.1 channels and the resulting outward current can stabilize the resting state of cells. Initial characterization of heterologously expressed human Slo2.1 indicated that these channels were inhibited by physiological levels of intracellular ATP. However, a subsequent study found that intracellular ATP had no effect on Slo2.1 channels. Here, we re-examine the effects of intracellular ATP on cloned human Slo2.1 channels heterologously expressed in Xenopus oocytes. Our studies provide both direct and indirect evidence that changes in intracellular [ATP] have no effect on Slo2.1 channels. First, we directly examined the effects of intracellular ATP on Slo2.1 channel activity in excised inside-out macropatches from Xenopus oocytes. Application of 5 mmol/L ATP to the intracellular solution did not inhibit Slo2.1 currents activated by niflumic acid. Second, we lowered the [ATP]i in whole oocytes using the metabolic inhibitor NaN3. Depletion of [ATP]i in oocytes by 3 mmol/L NaN3 rapidly activated heterologously expressed KATP channels, but did not increase wild-type Slo2.1 channel currents activated by niflumic acid or currents conducted by constitutively active mutant (E275D) Slo2.1 channels. Third, mutation of a conserved residue in the ATP binding consensus site in the C-terminal domain of the channel did not enhance the magnitude of Slo2.1 current as expected if binding to this site inhibited channel function. We conclude that Slo2.1 channels are not inhibited by intracellular ATP.
在正常生理条件下，Slo2.1钾离子通道的开放概率较低。缺血或细胞快速电起搏引起的胞质[Na⁺]和[Cl⁻]升高会增加Slo2.1通道的开放概率，由此产生的外向电流可稳定细胞的静息状态。对异源表达的人Slo2.1的初步表征表明，这些通道受到细胞内生理水平ATP的抑制。然而，随后的一项研究发现细胞内ATP对Slo2.1通道没有影响。在此，我们重新研究细胞内ATP对非洲爪蟾卵母细胞中异源表达的克隆人Slo2.1通道的影响。我们的研究提供了直接和间接证据，表明细胞内[ATP]的变化对Slo2.1通道没有影响。首先，我们直接检测了细胞内ATP对从非洲爪蟾卵母细胞切下的内面向外大膜片上Slo2.1通道活性的影响。向细胞内溶液中加入5 mmol/L ATP并未抑制尼氟灭酸激活的Slo2.1电流。其次，我们使用代谢抑制剂NaN₃降低了整个卵母细胞中的[ATP]i。3 mmol/L NaN₃使卵母细胞中的[ATP]i耗竭，迅速激活了异源表达的KATP通道，但并未增加尼氟灭酸激活的野生型Slo2.1通道电流或组成型活性突变体（E275D）Slo2.1通道传导的电流。第三，如果与该位点的结合抑制通道功能，那么通道C末端结构域中ATP结合共有位点的一个保守残基发生突变，并不会如预期那样增强Slo2.1电流的幅度。我们得出结论，Slo2.1通道不受细胞内ATP的抑制。
