Hill, A. D. | Search bioRxiv

bioRxiv2025

Measuring serotonin binding to its receptors via charge transfer to ANAP

Serotonin (5-HT) is a vital intercellular messenger with diverse signaling functions throughout the human body. We have characterized and implemented a novel, in vitro fluorescence-based method of measuring 5-HT binding to gain a fuller understanding of the interactions between 5- HT and its receptors. This method involves expression of 5-HT receptor proteins in cultured cells with the fluorescent, non-canonical amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2- aminopropanoic acid (ANAP) incorporated into the ligand binding site. ANAP fluorescence was quenched in solution by both 5-HT and dopamine. Time-resolved photoluminescence and transient absorption spectroscopy confirmed that ANAP quenching by 5-HT occurs via a charge- transfer process that recovers through back-electron transfer on the nanosecond timescale. Supported by density functional theory calculations, this process likely involved a ANAP reduction by 5-HT. To test this method on intact receptors in a cellular context, we expressed 5- HT3A receptors (5-HT-gated ion channels) in HEK293T cells with ANAP inserted co- translationally into the transmitter binding site. Fluorescently labeled 5-HT3A receptors were functional and activated by 5-HT, as assessed by whole-cell patch clamp. Addition of 5-HT caused a concentration-dependent quenching of fluorescence from ANAP-tagged channels in intact cells and unroofed plasma membranes, demonstrating the utility of this method for measuring 5-HT binding to its receptors. Collectively, these results delineate a technique for measuring transmitter binding that can be widely adopted to explore 5-HT binding not only to 5- HT3 receptors, but to any 5-HT receptor, transporter, or binding protein in heterologous expression systems.

biophysics↗

bioRxiv2021

Sensitivity of discrete symmetry metrics: implications for metric choice

Gait asymmetry is present in several pathological populations, including those with Parkinsons disease, Huntingtons disease, and stroke survivors. Previous studies suggest that commonly used discrete symmetry metrics, which compare single bilateral variables, may not be equally sensitive to underlying effects of asymmetry, and the use of a metric with low sensitivity could result in unnecessarily low statistical power. The purpose of this study was to provide a comprehensive assessment of the sensitivity of commonly used discrete symmetry metrics to better inform design of future studies. Monte Carlo simulations were used to estimate the statistical power of each symmetry metric at a range of asymmetry magnitudes, group/condition variabilities, and sample sizes. Power was estimated by repeated comparison of simulated symmetric and asymmetric data with a paired t-test, where the proportion of significant results is equivalent to the power. Simulation results confirmed that not all common discrete symmetry metrics are equally sensitive to reference effects of asymmetry. Multiple symmetry metrics exhibit equivalent sensitivities, but the most sensitive discrete symmetry metric in all cases is a bilateral difference (e.g. left - right). A ratio (e.g. left/right) has poor sensitivity when group/condition variability is not small, but a log-transformation produces increased sensitivity. Additionally, two metrics which included an absolute value in their definitions showed increased sensitivity when the absolute value was removed. Future studies should consider metric sensitivity when designing analyses to reduce the possibility of underpowered research. Summary statementStatistical power is an important factor in study design. Our results show that not all discrete symmetry metrics have similar or sufficient sensitivity to detect effects of asymmetry.

bioengineering↗