Diesch Ann-Kathrin, Grissmer Stephan
Cell Physiol Biochem. 2017;44(1):172-184. doi: 10.1159/000484625. Epub 2017 Nov 6.
BACKGROUND/AIMS: The human-voltage gated Kv1.3 channel (hKv1.3) is expressed in T- and B lymphocytes. Verapamil is able to block hKv1.3 channels. We characterized the effect of verapamil on currents through hKv1.3 channels paying special attention to the on-rate (kon) of verapamil. By comparing on-rates obtained in wild-type (wt) and mutant channels a binding pocket for verapamil and impacts of different amino acid residues should be investigated.
Using the whole-cell patch clamp technique the action of verapamil on currents through wild-type and six hKv1.3 mutant channels in the open state was investigated by measuring the time course of the open channel block in order to calculate kon of verapamil.
The on-rate of verapamil to block current through hKv1.3_T419C mutant channels is similar to that obtained for hKv1.3_wt channels whereas the on-rate of verapamil to block currents through hKv1.3_L417C and hKv1.3_L418C mutant channels was ∼ 3 times slower compared to in wt channels. The on-rate of verapamil to block currents through hKv1.3_L346C and the double mutant hKv1.3_L346C_L418C channel was ∼ 2 times slower compared to that obtained in the wt channel. The hKv1.3_I420C mutant channel reduced the on-rate of verapamil to block currents ∼ 6 fold.
We conclude that position 420 in hKv1.3 channels maximally interferes with verapamil reaching its binding site to block the channel. Positions 417 and 418 in hKv1.3 channels partially hinder verapamil reaching its binding site to block the channel whereas position 419 may not interfere with verapamil at all. Mutant hKv1.3_L346C and hKv1.3_L346C_L418C mutant channels might indirectly influence the ability of verapamil reaching its binding site to block current.
背景/目的：人类电压门控Kv1.3通道（hKv1.3）在T淋巴细胞和B淋巴细胞中表达。维拉帕米能够阻断hKv1.3通道。我们对维拉帕米对通过hKv1.3通道的电流的影响进行了表征，特别关注维拉帕米的结合速率（kon）。通过比较野生型（wt）和突变通道中获得的结合速率，应研究维拉帕米的结合口袋以及不同氨基酸残基的影响。
使用全细胞膜片钳技术，通过测量开放通道阻断的时间进程来研究维拉帕米对野生型和六个处于开放状态的hKv1.3突变通道电流的作用，以计算维拉帕米的kon。
维拉帕米阻断通过hKv1.3_T419C突变通道电流的结合速率与hKv1.3_wt通道的相似，而维拉帕米阻断通过hKv1.3_L417C和hKv1.3_L418C突变通道电流的结合速率比野生型通道慢约3倍。维拉帕米阻断通过hKv1.3_L346C和双突变hKv1.3_L346C_L418C通道电流的结合速率比野生型通道慢约2倍。hKv1.3_I420C突变通道使维拉帕米阻断电流的结合速率降低约6倍。
我们得出结论，hKv1.3通道中的420位最大程度地干扰了维拉帕米到达其结合位点以阻断通道。hKv1.3通道中的417位和418位部分阻碍维拉帕米到达其结合位点以阻断通道，而419位可能根本不干扰维拉帕米。突变的hKv1.3_L346C和hKv1.3_L346C_L418C突变通道可能间接影响维拉帕米到达其结合位点以阻断电流的能力。
