Biology subjects

Schmauder, R.

Publications and source records attributed to Schmauder, R..

5 recordsLinked to original sources

bioRxiv2024

Subunit-specific conductance of single HCN pacemaker channels at femtosiemens resolution

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetramers that generate rhythmic electrical activity in neuronal and cardiac pacemaker cells 1. The channels are activated by hyperpolarisation of the membrane voltage and additionally tuned by the second messenger cAMP at sympathetic stimulation. There are four mammalian isoforms, HCN1-4 2-4. The single-channel conductance,{gamma} , of HCN channels remains debated, with conflicting results ranging from near 1.5 pS for HCN2 5,6 to tens of pS for HCN1 7, HCN2 7 and HCN4 7,8, though the pore structure, viewed to determine the conductance 9,10, is either identical or highly conserved. To resolve this controversy, we analyzed all four mouse isoforms mHCN1-4 at femtosiemens resolution. We show that mHCN1, mHCN3 and mHCN4 also generate small conductance values, even smaller than that of mHCN2 with the sequence{gamma} mHCN2=1.54 pS >{gamma} mHCN1=0.84 pS >{gamma} mHCN3=0.54 pS {asymp}{gamma} mHCN4=0.51 pS. As shown by systematic mutagenesis and molecular dynamic simulations, the differences in the conductance are neither generated by the selectivity filter nor the inner gate 9, but by defined negative charges in the outer channel vestibule increasing cation occupation. In line with these results, heteromers of mHCN2 with either mHCN1, mHCN3 or mHCN4 lead to graded single-channel currents in-between those of the respective homomeric channels. Our approach at femtosiemens resolution provides insight into the function of recombinant and native HCN channels at the level of single subunits and is thus promising for the development of subunit-specific drugs acting on these clinically highly relevant channels 11.

physiology↗

bioRxiv2024

Minimally and vaguely informative priors to combat practical parameter non-identifiability of hidden Markov models exemplified by ion channel data

Hidden Markov models (HMMs) for biomolecules suffer from various forms of parameter non-identifiability. This poses severe challenges to both maximum likelihood and Bayesian inference. However, Bayesian inference offers effective means of overcoming these pathologies. We study the role of prior distributions in the face of practical parameter non-identifiability in Bayesian inference applied to prototypical patch clamp data of ligand-gated ion channels. We advocate the use of minimally informative priors, as they increase the accuracy and decrease the uncertainty of the inference. For complex HMMs, stronger prior assumptions are needed to render the posterior sufficiently proper. This can be achieved by confining the parameter space to physically motivated limits. Another beneficial assumption is finite cooperativity of ligand-binding and unbinding events, which introduces a bias towards non-cooperativity but still allows for a non-vanishing degree of cooperativity that is inferred from the data. Despite its vagueness, our prior renders the posterior sufficiently proper for all datasets that we considered without imposing the assumption of non-cooperativity. Combining all prior factors allows for meaningful inferences with a dataset of a thousand times lower quality.

bioinformatics↗

bioRxiv2023

cAMP binding to closed pacemaker ion channels is cooperative

Most of receptor proteins in biomembranes are oligomers of either homologue or identical subunits, that govern a cooperative activation process. HCN ion channels, generating electric rhythmicity in specialized brain neurons and cardiomyocytes1, are tetramers activated by hyperpolarizing voltage and modulated by the intracellular binding of the second messenger cAMP2,3. The molecular processes underlying the cooperative action of the subunits4-6 by the binding are not fully understood, in particular if they are necessarily associated with channel activation7 or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides8. Here we show positive cooperativity in ligand binding to resting HCN2 channels in native cell membranes by analyzing the binding of fluorescence-labelled single cAMP derivatives to the channels. Kinetic modelling reveals, that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in resting HCN2 channels is already cooperative prior to channel activation. This shows that single-molecule binding studies can quantify cooperativity in ligand binding to receptors in native membranes based on equilibrium measurements.

biophysics↗

bioRxiv2020

Functional and structural characterization of interactions between opposite subunits in HCN pacemaker channels

Hyperpolarization-activated and cyclic nucleotide (HCN) modulated channels are tetrameric cation channels. In each of the four subunits, the intracellular cyclic nucleotide-binding domain (CNBD) is coupled to the transmembrane domain via a helical structure, the C-linker. High-resolution channel structures suggest that the C-linker enables functionally relevant interactions with the opposite subunit, which might be critical for coupling the conformational changes in the CNBD to the channel pore. We combined mutagenesis, patch-clamp technique, confocal patch-clamp fluorometry, and molecular dynamics simulations to show that residue K464 of the C-linker is essential for stabilizing the closed state of the mHCN2 channel by forming interactions with the opposite subunit. MD simulations revealed that both cAMP and K464E induce a rotation of the intracellular domain relative to the channel pore, weakening the autoinhibitory effect of the unoccupied CL-CNBD region. The adopted poses are in excellent agreement with structural results.

biophysics↗

bioRxiv2020

Bayesian Hidden Markov modeling and model selection by Kalman filtering applied to multi-dimensional data of ion channels

Inferring adequate kinetic schemes for ion channel gating from ensemble currents is a daunting task due to limited information in the data. We address this problem by using a parallelized Bayesian filter to specify hidden Markov models for current and fluorescence data. We demonstrate the flexibility of this algorithm by including different noise distributions. Our generalized Kalman filter outperforms both a classical Kalman filter and a rate equation approach when applied to patch-clamp data exhibiting realistic open-channel noise. The derived generalization also enables inclusion of orthogonal fluorescence data, making unidentifiable parameters identifiable and increasing the accuracy of the parameter estimates by an order of magnitude. By using Bayesian highest credibility volumes, we found that our approach, in contrast to the rate equation approach, yields a realistic quantification of uncertainty. Furthermore, the Bayesian filter delivers unbiased estimates for a wider range of data quality and identifies parameters which the rate equation approach does not identify. These results also demonstrate the power of assessing the validity of algorithms by Bayesian credibility volumes in general. Finally, we show that our Bayesian filter is more robust against errors induced by either analog filtering before analog-to-digital conversion or by limited time resolution of fluorescence data than a rate equation approach.

bioinformatics↗