Li Kangjun, Janve Vaishali Satpute, Denton Jerod S
Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee, United States.
Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, United States.
Am J Physiol Cell Physiol. 2025 Jul 1;329(1):C82-C92. doi: 10.1152/ajpcell.00266.2025. Epub 2025 Jun 4.
ATP-sensitive potassium (K) channels are therapeutic targets for numerous metabolic, cardiovascular, and neurological disorders. Drug development for K channels requires electrophysiology assays for detailed compound characterization. Parallel automated patch clamp (APC) techniques offer considerable advantages over low-throughput manual patch clamp electrophysiology. Here, we characterized the functional properties and pharmacological sensitivity of heterologously expressed Kir6.2/SUR1 and Kir6.1/SUR2B using a SyncroPatch 384PE APC instrument. Ruptured-membrane and perforated-patch whole cell recordings in potassium fluoride and fluoride-free assay buffers and electrophysiology chips were evaluated for both subtypes. Effects of internal ATP and ADP, and magnesium (Mg) addition were also assessed. Kir6.2/SUR1 currents were constitutively active in all potassium fluoride-based recordings, insensitive to activation by the SUR1 agonist, VU0071063, and variably inhibited by glibenclamide. Success rates, current rundown, and glibenclamide sensitivity were associated with internal buffer composition. Recordings in fluoride-free buffers revealed a minor population of constitutively active Kir6.2/SUR1 currents and a larger population of currents exhibiting low basal activity and activation by VU0071063. Success rate and stability were associated with internal buffer composition. Kir6.1/SUR2B currents, which were most readily assayed in ruptured-membrane and potassium fluoride-based conditions, were stable, activatable with pinacidil, and inhibited by glibenclamide. Our study sheds new light on the behavior of Kir6.2/SUR1 and Kir6.1/SUR2B currents under available APC conditions and represents an important step toward developing truly high-throughput APC techniques for K. Highly parallel automated patch clamp (APC) methods have revolutionized the way electrophysiology is performed in the pharmaceutical and biotechnology industries and increasingly in academic laboratories. Here, we characterized the functional and pharmacological properties of heterologously expressed Kir6.2/SUR1 and Kir6.1/SUR2B using a SyncroPatch 384PE APC instrument. The results of our studies highlight heretofore unappreciated effects of fluoride-base internal solutions on Kir6.2/SUR1 and provide foundational support for developing truly high-throughput electrophysiology methods for both drug targets.
ATP敏感性钾（K）通道是多种代谢、心血管和神经疾病的治疗靶点。开发针对K通道的药物需要进行电生理测定以详细表征化合物。与低通量手动膜片钳电生理相比，并行自动膜片钳（APC）技术具有显著优势。在此，我们使用SyncroPatch 384PE APC仪器对异源表达的Kir6.2/SUR1和Kir6.1/SUR2B的功能特性和药理敏感性进行了表征。针对这两种亚型，评估了在氟化钾和无氟测定缓冲液以及电生理芯片中进行的破膜和穿孔膜全细胞记录。还评估了内部ATP和ADP以及添加镁（Mg）的影响。在所有基于氟化钾的记录中，Kir6.2/SUR1电流呈组成性激活，对SUR1激动剂VU0071063的激活不敏感，并受到格列本脲的不同程度抑制。成功率、电流衰减和格列本脲敏感性与内部缓冲液组成有关。在无氟缓冲液中的记录显示，一小部分Kir6.2/SUR1电流呈组成性激活，而更大比例的电流表现出低基础活性并可被VU0071063激活。成功率和稳定性与内部缓冲液组成有关。Kir6.1/SUR2B电流在破膜和基于氟化钾的条件下最易于检测，其电流稳定，可被吡那地尔激活，并受到格列本脲抑制。我们的研究为在现有APC条件下Kir6.2/SUR1和Kir6.1/SUR2B电流的行为提供了新的见解，代表了朝着开发真正高通量的K通道APC技术迈出的重要一步。高度并行的自动膜片钳（APC）方法彻底改变了制药和生物技术行业以及越来越多学术实验室中进行电生理研究的方式。在此，我们使用SyncroPatch 384PE APC仪器对异源表达的Kir6.2/SUR1和Kir6.1/SUR2B的功能和药理特性进行了表征。我们的研究结果突出了迄今为止未被重视的基于氟化物的内部溶液对Kir6.2/SUR1的影响，并为开发针对这两种药物靶点的真正高通量电生理方法提供了基础支持。
