Electrophysiological studies investigating drug effects on hHCN1 channels and Unravelling pore dynamics in Pannexin channels

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel stands out for its unique characteristics, including sensitivity to transmembrane voltage and cyclic nucleotides, as well as its non-selective passage of monovalent cations. These channels play pivotal roles in cardiac rhythmicity, dendritic integration, memory, etc. Reports indicate potential modulation of HCN channels by acetylcholine via muscarinic acetylcholine receptors, both indirectly and directly. Furthermore, studies suggest that nicotine exhibits a blocking effect on HCN channels in OLM interneurons, displaying greater potency than the known blocker ZD7288. However, these investigations were conducted in vitro on neurons without knowledge of the specific HCN isoform distribution profile, making it challenging to ascertain isoform specificity. To address these limitations, we examined the effects of nicotine and acetylcholine on hHCN1 isoform using patch clamp electrophysiology by expressing them in HEK293 cells, eliminating interference from other HCN isoforms and ion. Our electrophysiology studies did not reveal nicotine and acetylcholine as potent blockers of HCN channels; however, nicotine significantly slowed the activation kinetics of hHCN1 channels. Continuing our investigation, we shifted our focus towards identifying FDA-approved drugs that could be repurposed to effectively block HCN channels. We streamlined our selection by first identifying FDA-approved drugs that cause bradycardia. Then, we compared their molecular structures to a template generated using potent HCN blockers, leading to eight promising compounds for further study. We subjected the shortlisted compounds to the molecular docking studies using the modelled open conformation homology model of the pore of the hHCN1 channel generated using modeller. Among the shortlisted drugs, donepezil emerged as a prime candidate for our electrophysiology study due to its favorable binding energy, established neuroprotective effects, and structural similarity to ivabradine. Electrophysiology experiments revealed that donepezil effectively blocks HCN channels. We further studied the voltage dependent modulation of hHCN1 channel kinetics by donepezil, blockade properties and dose response. We also identified the residues of hHCN1 channels involved in the interaction with donepezil by incorporating point mutations. In the final part of the thesis, we studied human Pannexin channels, focusing on two key isoforms, PANX1 and PANX3, along with their respective mutants. Our aim was to gain insights into the pore region of these channels by comparing electrophysiological findings with structural variations arising from isoform differences or mutations. Our study marks the first reporting of electrophysiology recordings from PANX3 channels, contributing novel insights into their functional characteristics. We also created a double mutant of PANX1 channels designed to replicate the pore lining residues found in PANX2 channels. Through comparative electrophysiological analysis, we elucidated differences in the behavior of these mutants. Further, we investigated the mutation of I74 in PANX3, a residue critical for creating the narrowest point in the pore, by substituting it with charged and neutral amino acids. Moreover, our study involved electrophysiological investigations on C-terminus deleted PANX3 variants, to investigate the role of this region in channel activity. In addition, the functional consequences of the germline mutant R217H in PANX1 were compared to wild-type PANX1. Finally, we examined mutations in PANX1 channel pore known for their roles in ATP release and ion transport, elucidating their functional significance.