Review

Genetic manipulation of ion channels: A new approach to structure and mechanism

Conventional site-directed mutagenesis and genetic code expansion approaches have been instrumental in providing detailed functional and pharmacological insight into membrane proteins such as ion channels. Recently, this has increasingly been complemented by semi-synthetic strategies, in which part of the protein is generated synthetically. This means a vast range of chemical modifications, including non-canonical amino acids (ncAA), backbone modifications, chemical handles, fluorescent or spectroscopic labels and any combination of these can be incorporated. Among these approaches, protein trans-splicing (PTS) is particularly promising for protein reconstitution in live cells. It relies on one or more split inteins, which can spontaneously and covalently link flanking peptide or protein sequences. Here, we describe the use of PTS and its variant tandem PTS (tPTS) in semi-synthesis of ion channels in Xenopus laevis oocytes to incorporate ncAAs, post-translational modifications or metabolically stable mimics thereof. This strategy has the potential to expand the type and number of modifications in ion channel research.

Nicotinic acetylcholine receptors (nAChR) are homo- or hetero-pentameric ligand-gated ion channels of the Cys-loop superfamily and play important roles in the nervous system and muscles. Studies on nAChR benefit from in silico modeling due to the lack of high-resolution structures for most receptor subtypes and challenges in experiments addressing the complex mechanism of activation involving allosteric sites. Although there is myriad of computational modeling studies on nAChR, the multitude of the methods and parameters used in these studies makes modeling nAChR a daunting task, particularly for the non-experts in the field. To address this problem, the modeling literature on Torpedo nAChR and α7 nAChR were focused on as examples of heteromeric and homomeric nAChR, and the key in silico modeling studies between the years 1995–2019 were concisely reviewed. This was followed by a critical analysis of these studies by comparing the findings with each other and with the emerging experimental and computational data on nAChR. Based on these critical analyses, suggestions were made to guide the future researchers in the field of in silico modeling of nAChR.

This article is part of the special issue on ‘Contemporary Advances in Nicotine Neuropharmacology’.

Fenamates mefanamic and niflumic acids (MFA and NFA) induced dual potentiating and inhibitory effects on GABA currents recorded in isolated cerebellar Purkinje cells using the whole-cell patch-clamp and fast-application techniques. Regardless of the concentration, both drugs induced a pronounced prolongation of the current response. We demonstrated that the same concentration of drugs can produce both potentiating and inhibitory effects, depending on the GABA concentration, which indicates that both processes take place simultaneously and the net effect depends on the concentrations of both the agonist and fenamate. We found that the NFA-induced block is strongly voltage-dependent. The Woodhull analysis of the block suggests that NFA has two binding sites in the pore - shallow and deep. We built a homology model of the open GABA_AR based on the cryo-EM structure of the open α1 GlyR and applied Monte-Carlo energy minimization to optimize the ligand-receptor complexes. A systematic search for MFA/NFA binding sites in the GABA_AR pore revealed the existence of two sites, the location of which coincides well with predictions of the Woodhull model. In silico docking suggests that two fenamate molecules are necessary to occlude the pore. We showed that MFA, acting as a PAM, competes with an intravenous anesthetic etomidate for a common binding site. We built structural models of MFA and NFA binding at the transmembrane β(+)/α(−) intersubunit interface. We suggested a hypothesis on the molecular mechanism underlying the prolongation of the receptor lifetime in open state after MFA/NFA binding and β subunit specificity of the fenamate potentiation.

Structural rearrangements underlying functional transitions of pentameric ligand-gated ion channels (pLGICs) are not fully understood. Using ¹⁹F nuclear magnetic resonance and electron spin resonance spectroscopy, we found that ELIC, a pLGIC from Erwinia chrysanthemi, expanded the extracellular end and contracted the intracellular end of its pore during transition from the resting to an apparent desensitized state. Importantly, the contraction at the intracellular end of the pore likely forms a gate to restrict ion transport in the desensitized state. This gate differs from the hydrophobic gate present in the resting state. Conformational changes of the TM2-TM3 loop were limited to the N-terminal end. The TM4 helices and the TM3-TM4 loop appeared relatively insensitive to agonist-mediated structural rearrangement. These results indicate that conformational changes accompanying functional transitions are not uniform among different ELIC regions. This work also revealed the co-existence of multiple conformations for a given state and suggested asymmetric conformational arrangements in a homomeric pLGIC.

As one of the most important predatory enemies, the miridbug, Cyrtorhinus lividipennis, plays an important role in rice planthoppers control, such as Nilaparvata lugens (brown planthopper). In order to compare insecticide selectivity between C. lividipennis and N. lugens, the contact acute toxicities of six insecticides (diazoxon, paraoxon, carbaryl, fenobucarb, fipronil and ethofenprox) were monitored. The results showed that all tested insecticides were more toxic to C. lividipennis than to N. lugens and fipronil had the biggest difference. The RDL subunit (Cl-RDL) was cloned from C. lividipennis and a RDL isoform (Cl-RDL-In) was also found with 31 amino acids insertion in RDL intracellular region. In order to understand the role of the insertion on insecticide sensitivities, three subunits (Nl-RDL, Cl-RDL and Cl-RDL-In) were constructed to obtain the functional receptors in Xenopus oocytes and the fipronil sensitivities were detected by the voltage-clamp technique. Nl-RDL (IC₅₀=32.36 ± 4.07 µM) was more insensitive to fipronil than Cl-RDL (IC₅₀=6.47 ± 1.12 µM). The insertion in Cl-RDL significantly reduced fipronil sensitivity with IC₅₀ value in Cl-RDL-In of 16.83 ± 2.30 µM. Interestingly, after the elution of fipronil, the current response of Cl-RDL-In appeared obvious recovery, which were not observed in Cl-RDL and Nl-RDL. It might imply that the insertion played a special role in fipronil sensitivity.

Nicotinic acetylcholine receptors (nAChR's) containing the α6 subunit (α6^⁎) are putative drug targets of relevance to Parkinson's disease and nicotine addiction. However, heterologous expression of α6^⁎ receptors has proven challenging which has stifled drug discovery efforts. Here, we investigate potential new avenues for achieving functional α6^⁎ receptor expression. Combinations of chimeric and mutated α6, β2 and β3 subunits were co-expressed in the human HEK293 cell line and receptor expression was assessed using Ca²⁺-imaging (FLIPR™) and whole-cell patch-clamp electrophysiology. Transient transfections of a chimeric α6/α3 subunit construct in combination with β2 and β3^V9'S gave rise to significant acetylcholine-evoked whole-cell currents. Increasing the β3^V9'S:β2:α6/α3 cDNA ratio, resulted in a significantly higher fraction of cells with robust current levels. Using an excess of wild-type β3, significant functional expression of α6/α3β2β3 was also demonstrated. Comparing the acetylcholine concentration–response relationship of α6/α3β2β3^V9'S to that of α6/α3β2β3 revealed the β3 point mutation to result in decreased current decay rate and increased ACh agonist potency. Ca²⁺-imaging experiments showed preservation of basic α6^⁎ receptor pharmacology. Our results establish that α6/α3β2β3^V9'S replicate several basic features of native α6^⁎ receptors but also highlight several caveats associated with using this construct and may therefore provide guidance for future drug hunting efforts.