5-Chloro-2-Guanidinobenzimidazole (ClGBI) Is a Non-Selective Inhibitor of the Human H_V1 Channel

5-chloro-2-guanidinobenzimidazole (ClGBI), a small-molecule guanidine derivative, is a known effective inhibitor of the voltage-gated proton (H⁺) channel (H_V1, K ≈ 26 μM) and is widely used both in ion channel research and functional biological assays. However, a comprehensive study of its ion channel selectivity determined by electrophysiological methods has not been published yet. The lack of selectivity may lead to incorrect conclusions regarding the role of hHv1 in physiological or pathophysiological responses in vitro and in vivo. We have found that ClGBI inhibits the proliferation of lymphocytes, which absolutely requires the functioning of the K_V1.3 channel. We, therefore, tested ClGBI directly on hK_V1.3 using a whole-cell patch clamp and found an inhibitory effect similar in magnitude to that seen on hH_V1 (K ≈ 72 μM). We then further investigated ClGBI selectivity on the hK_V1.1, hK_V1.4-IR, hK_V1.5, hK_V10.1, hK_V11.1, hK_Ca3.1, hNa_V1.4, and hNa_V1.5 channels. Our results show that, besides H_V1 and K_V1.3, all other off-target channels were inhibited by ClGBI, with K values ranging from 12 to 894 μM. Based on our comprehensive data, ClGBI has to be considered a non-selective hH_V1 inhibitor; thus, experiments aiming at elucidating the significance of these channels in physiological responses have to be carefully evaluated.