Biology subjects

Hotka, M.

Publications and source records attributed to Hotka, M..

4 recordsLinked to original sources

bioRxiv2025

IP3-mediated Ca2+ transfer from ER to mitochondria stimulates ATP synthesis in primary hippocampal neurons

During electrical activity, Ca2+ enhances mitochondrial ATP production, helping to replenish the energy consumed during this process. Most Ca2+ enters the cell via ligand- or voltage-gated channels on the neuronal membrane, where it stimulates the release of additional Ca2+ from the endoplasmic reticulum (ER). Although the influence of cytosolic Ca2+ on neuronal metabolism has been widely investigated, relatively few studies have explored the contribution of ER Ca2+ release in this context. Therefore, we investigated how activity-driven Ca2+ crosstalk between the ER and mitochondria influences the regulation of mitochondrial ATP production. We show that in primary hippocampal neurons derived from rat pups of either sex, depletion of ER Ca2+ led to a reduction in mitochondrial Ca2+ levels during both resting and stimulated states, while exerting only a minimal impact on cytosolic Ca2+ levels. Additionally, impaired ER-mitochondria Ca{superscript 2} transfer led to a reduction in mitochondrial ATP production. Similar effects were observed when inositol-3-phosphate receptors (IP3Rs), but not ryanodine receptors (RyRs), were pharmacologically inhibited. Together, our findings show that, in hippocampal neurons, Ca2+ is transferred from the ER to mitochondria through IP3 receptors, and this Ca2+ crosstalk in turn enhances mitochondrial ATP production in response to neuronal activity. HighlightsO_LICa2+ adjusts mitochondrial ATP synthesis to neuronal activity C_LIO_LIIn the neuronal somata ER-mitochondria Ca2+ crosstalk occurs via IP3 receptors C_LIO_LIIP3-mediated Ca2+ release occurs across a wide range of firing intensities. C_LI

neuroscience↗

bioRxiv2019

An electrophysiological approach to measure changes in the membrane surface potential in real time

Biological membranes carry fixed charges at their surfaces. These arise primarily from phospholipid head groups. In addition, membrane proteins contribute to the surface potential with their charged residues. Membrane lipids are asymmetrically distributed. Because of this asymmetry the net negative charge at the inner leaflet exceeds that at the outer leaflet. Changes in surface potential are predicted to shape the capacitive properties of the membrane (i.e. the ability of the membrane to store electrical charges). Here, we show that it is possible to detect changes in surface potential by an electrophysiological approach: the analysis of cellular currents relies on assuming that the electrical properties of a cell are faithfully described by a three-element circuit - i.e. the minimal equivalent circuit - comprised of two resistors and one capacitor. However, to account for changes in surface potential it is necessary to add a battery to this circuit connected in series with the capacitor. This extended circuit model predicts that the current response to a square-wave voltage pulse harbors information, which allows for separating the changes in surface potential from a true capacitance change. We interrogated our model by investigating changes in capacitance induced by ligand binding to the serotonin transporter (SERT) and to the glycine transporters (GlyT1 and GlyT2). The experimental observations were consistent with the predictions of the extended circuit. We conclude that ligand-induced changes in surface potential (reflecting the binding event) and in true membrane capacitance (reflecting the concomitant conformational change) can be detected in real time even in instances where they occur simultaneously.\n\nStatement of SignificanceThe plasma membrane of a cell possesses fixed charges on both surfaces. Surface charges play an important role in many biological processes. However, the mechanisms, which regulate the surface charge densities at the plasma membrane, are poorly understood. This is in part due to lack of experimental approaches that allow for detecting changes in surface charges in real time. Here, we show that it is possible to track alterations in the electric potential at the membrane surface with high temporal resolution by an electrophysiological approach. Importantly, the described method allows for discriminating between a change in surface potential and a change in true membrane capacitance (e.g. a change in membrane area), even if these occur in parallel.

biophysics↗

bioRxiv2018

A label-free approach to detect ligand binding to cell surface proteins in real time

Electrophysiological recordings allow for monitoring the operation of proteins with high temporal resolution down to the single molecule level. This technique has been exploited to track either ion flow arising from channel opening or the synchronized movement of charged residues and/or ions within the membrane electric field. Here, we describe a novel type of current by using the serotonin transporter (SERT) as a model. We examined transient currents elicited on rapid application of specific SERT inhibitors. Our analysis shows that these currents originate from ligand binding and not from a conformational change. The Gouy-Chapman model predicts that a ligand-induced elimination/neutralization of surface charge must produce a displacement current and related apparent changes in membrane capacitance. Here we verified these predictions with SERT. Our observations demonstrate that ligand binding to a protein can be monitored in real time and in a label-free manner by recording the membrane capacitance.

biophysics↗

bioRxiv2017

Deconvolution of membrane currents for low-crosstalk high-resolution membrane capacitance measurements

Many cellular activities involving cell membrane manifest through changes in membrane capacitance. The accuracy and precision of cell membrane capacitance measurement is limited by the measuring system, which distorts membrane current responses used for analysis of impedance parameters. We developed a practical deconvolution procedure for reconstruction of the time course of membrane current and applied it to current responses recorded at step voltage changes by the whole-cell patch-clamp technique. In contrast to the recorded current responses, the reconstructed current responses could be exactly described by adequate impedance models of the recorded circuit. Deconvolution of membrane currents improved the performance of the square-wave method of high-resolution membrane capacitance recording by providing higher accuracy in a wider range of cell sizes and by eliminating cross-talk errors in parameter estimates. This allowed resolving instabilities in the recording conditions arising from parasitic capacitance and seal resistance variation. Complex tests on hardware models, on simulated data sets, and on living cells confirmed reliability of the deconvolution method for membrane capacitance recording. The aptitude of the method was demonstrated in isolated cardiac myocytes by recording spontaneous vesicular events, by discerning formation of a fusion pore, and by revealing artefacts due to unstable seal resistance.

biophysics↗