Biology subjects

Puljung, M. C.

Publications and source records attributed to Puljung, M. C..

3 recordsLinked to original sources

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↗

bioRxiv2025

A fluorescent probe for the enzymatic activity of KATP

Neuroendocrine ATP-sensitive K+ channels (KATP) comprise four pore-forming subunits (Kir6.2), each associated with a modulatory sulfonylurea receptor subunit (SUR1). ATP/ADP binding to Kir6.2 inhibits KATP; MgATP/MgADP binding to two different sites on SUR1 promotes activation. As SUR1 is a member of the ABC transporter family of proteins, it can potentially hydrolyze MgATP to MgADP. Whether this activity is required for KATP activation remains controversial. Previous studies demonstrated that non-hydrolyzable ATP analogs do not activate KATP, which may reflect an inability of these compounds to bind to SUR1, their inability to promote a conformational change in SUR1 that leads to channel activation, or a requirement for ATP hydrolysis during channel gating. To explore this further, we synthesized a fluorescent trinitrophenyl (TNP) derivative of the non-hydrolyzable ATP analog {beta},{gamma}-methyleneadenosine 5- triphosphate (AMP-PCP). Synthesis was verified by UV-visible absorbance, fluorescence spectroscopy, 1H nuclear magnetic resonance, and mass spectrometry. Purity was assessed by reversed-phase high-performance liquid chromatography. Real-time nucleotide binding to intact KATP channels in cell membranes was measured using FRET between channels labeled with a fluorescent, non-canonical amino acid and TNP-nucleotide derivatives. This technique provides sufficient spatial resolution to discriminate between binding to each site on KATP. Using this approach we first established that TNP-ATP can bind to nucleotide binding site 1 on SUR1 in fluorescently labeled Kir6.2/SUR1 channels in unroofed membranes of HEK293T cells. We subsequently demonstrated that TNP-AMP-PCP binds to both nucleotide binding sites on SUR1 in the absence of Mg2+. AMP-PCP was able to compete with TNP-ATP for binding to NBS2, suggesting that it, too, binds NBS2. We conclude that the failure of non-hydrolyzable ATP analogs to activate KATP does not stem from an inability of these nucleotides to bind to the channel.

biophysics↗

bioRxiv2019

Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry

Pancreatic ATP-sensitive K+ channels (KATP) comprise four inward rectifier subunits (Kir6.2), each associated with a sulphonylurea receptor (SUR1). ATP/ADP binding to Kir6.2 shuts KATP. Mg-nucleotide binding to SUR1 stimulates KATP. In the absence of Mg2+, SUR1 increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We simultaneously measured channel currents and nucleotide binding to Kir6.2. Fits to combined data sets suggest that KATP closes with only one nucleotide molecule bound. A Kir6.2 mutation (C166S) that increases channel activity did not affect nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. Mutations at position K205 in SUR1 affected both nucleotide affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in directly contributing to nucleotide binding and in stabilising the nucleotide-bound closed state.

biophysics↗