Acetylcholine receptors (AChRs) are members of a superfamily of proteins called pentameric ligand-gated ion channels, which are found in almost all forms of life and thus have a rich evolutionary history. Muscle-type AChRs are heteropentameric complexes assembled from four related subunits (α, β, δ, and ɛ). Here we reconstruct the amino acid sequence of a β subunit ancestor shared by humans and cartilaginous fishes (i.e., Torpedo). Then, by resurrecting this ancestral β subunit and co-expressing it with human α, δ, and ɛ subunits, we show that despite 132 substitutions, the ancestral subunit is capable of forming human/ancestral hybrid AChRs. Whole-cell currents demonstrate that the agonist acetylcholine has reduced potency for hybrid receptors, while single-channel recordings reveal that hybrid receptors display reduced conductance and open probability. Our results outline a promising strategy for studies of AChR evolution aimed at identifying the amino acid origins of AChR structure and function.
Recently, a couple of giga-seal automated patch clamp (APC) platforms in a standard 384-well plate format have been introduced. These APC instruments can deliver higher throughput measurements with high-quality data for ion channel drug discovery. In this study, we developed an automated electrophysiology assay for the voltage-gated potassium channel, Kv1.3 as a test case on the Nanion SyncroPatch 384PE, one of the latest APC platforms. We achieved a high cell-catch rate (98% wells/plate, defined by membrane resistance >16 MU/cell) by screening plate types (number of holes and resistance), and obtained a stable seal (87% wells/plate, defined by initial/final seal resistance >500 MU/cell) by optimizing recording solutions. We generated data at an overall success rate (defined by initial peak current >300 pA/cell, initial/final seal resistance >500 MU/cell, and current stability <57% change/min) of 79% on average. Using the same platform, we validated a dose-response assay using clotrimazole, a potent Kv1.3 inhibitor. The assay was robust with Z' factor 0.52 and the success rate 75%. Altogether, our results demonstrated that the SyncroPatch represents a reliable platform for voltage-gated potassium channel assays.
Nucleic acid extraction is the first step in molecular biology studies of soil bacterial communities. The most common used soil DNA extraction method is the direct, hard extraction Mobio method, which uses bead beating to lyse bacteria. In this study we compared the Mobio method with a soft, enzymatic lysis extraction method. Next generation sequencing (Illumina and Pyrosequencing) of amplicons generated from four 16S primer pairs and DNA from 12 soils and 3 composts was used to compare the two extraction methods. Four bacterial orders, the delta proteobacterial Desulfuromonadales and gamma proteobacterial Pseudomonadales, Enterobacteriales, and Alteromonadales were more common in amplicons from soft extracted DNA, sometimes by two orders of magnitude. These groups can be a significant fraction of the bacterial population. For example the Pseudomonadales made up to 16 % and Enterobacteriales 10% of amplicons from Soft extracted DNA. The JG30-KF-CM45 order was under extracted by the enzymatic lysis extraction method. Results differed more by primer choice than extraction method and the phylogenetic resolution of differences between extraction methods changed with primer choice. Given how often Mobio extraction is used, these proteobacterial orders are probably under-represented in the studies of soil bacteria that use nucleic acid methods. Further improvements in soil DNA extraction are needed. Amplicons sequencing studies should use a range of different primers to confirm the phylogenetic resolution of their results.
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