Identification of previously unknown ion channels is always a challenging work though the transmembrane protein-coding genes in human genome has been comprehensively annotated for years. Despite being the gold standard of functional assay for ion channels, electrophysiological recordings are often accompanied by electrical noise, leak conductance and background currents of the membrane system. These unwanted signals, if not treated properly, lead to mischaracterization of proteins with seemingly unusual ion-conducting properties. In recent ten years, the technical revolution of cryo-electron microscopy (cryo-EM) have greatly advanced our understanding on the structures and gating mechanisms of various ion channels, and also raised concerns about the pore-forming ability of some previously identified channel proteins. In this review, we summarize cryo-EM findings on ion channels with molecular identities recognized or disputed in recent ten years, and discuss current knowledge of proposed channel proteins awaiting cryo-EM analyses. We also present a classification of ion channels according to their architectures and evolutionary relationships, and discuss the possibility and strategy of identifying more ion channels by analyzing structures of transmembrane proteins of unknown function.

Introduction GFA (Guan-Fu Base A) as one of the main active substances in the Chinese medicine Ranunculaceae Aconite, has been approved the effect of anti-atrial fibrillation via its atrial-selective Na + channel-blocking action. It is recently undergoing phase IV clinical study. However, the molecular mechanism of Na V1.5 channel inhibition by GFA is largely unclear. Methods and Results Na V1.5 channel and its mutants were expressed in Xenopus oocytes and the currents were recorded with two-microelectrode voltage-clamp. GFA inhibited Na V1.5 currents in a concentration-dependent manner, with IC 50 of 66.24 μM, 371.59 μM, and 381.08 μM for wild type (WT), Delta KPQ (∆KPQ) and R1623Q constructs, respectively. Both the mutations of ∆KPQ and R1623Q decreased inhibitory potency of GFA about 5~6-fold. N406K mutation significantly altered the inhibition effect of GFA. Even 1 mM GFA has almost no inhibitory effect on the mutant. For both the WT and mutant channels, GFA reduced the currents in concentration, voltage and time dependent manner. Conclusion: GFA is a potent blocker of Na V1.5 channel. N406, the aromatic residues in the transmembrane helical of DIS6, is most likely responsible for the high-affinity binding of GFA to Na V1.5 channel.