Biology subjects

Benndorf, K.

Publications and source records attributed to Benndorf, K..

7 recordsLinked to original sources

bioRxiv2025

Selectivity Filter Dynamics Define Ion Conductance and Selectivity Differences in CNG and HCN Channels

Cyclic nucleotide-gated (CNG) channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are key members of the cyclic nucleotide-activated ion channel family that translate intracellular cyclic nucleotide binding into electrical signals. Functionally, CNG channels drive large inward currents in photoreceptors and olfactory sensory neurons, whereas HCN channels are best known for their roles in pacemaker activity in the heart and the regulation of neuronal excitability. Despite their considerable sequence similarity and conserved overall architecture, these channels exhibit striking differences in ion conductance, K+ selectivity, and voltage dependence. Here, we performed microsecond-timescale atomistic molecular dynamics (MD) simulations to directly compare the ion conduction mechanisms of HCN and CNG channels, using the prototypical K+-selective channel MthK as a reference. Our simulations reproduced key features observed in single-channel patch-clamp electrophysiology and revealed that distinct selectivity filter architectures and dynamic behaviors are the primary determinants underlying the divergence in ion conductance and K+ selectivity between HCN and CNG channels. Together, these results provide a mechanistic framework for understanding the physiological roles of these channels and pave the way for the rational design of cation channels with tailored functional properties.

biophysics↗

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

Functional properties of a disease mutation for migraine in Kv2.1/6.4 channels

Voltage-gated potassium (Kv) channels are integral to cellular excitability, impacting the resting membrane potential, repolarization, and shaping action potentials in neurons and cardiac myocytes. Structurally, Kv channels are homo or heterotetramers comprising four -subunits, each with six transmembrane segments (S1-S6). Silent Kv (KvS), includes Kv5.1, Kv6.1-6.4, Kv8.1-8.2, and Kv9.1-9.3, they do not form functional channels on their own but modulate the properties of heteromeric channels. Recent studies have identified the Kv6.4 subunit as a significant modulator within heteromeric channels, such as Kv2.16.4. The Kv2.16.4 heteromer exhibits altered biophysical properties, including a shift in voltage-dependent inactivation and a complex activation. Current genetic studies in migraine patients have revealed a single missense mutation in the Kv6.4 gene. The single missense mutation, L360P is in the highly conserved S4-S5 linker region. This study aims to demonstrate the biophysical effects of the L360P mutation in Kv2.1 6.4 channels with a fixed 2:2 stoichiometry, using monomeric (Kv2.1/6.4) and tandem dimer (Kv2.1_6.4) configurations. Our findings suggest that the L360P mutation significantly impacts the function and regulation of Kv2.1/6.4 channels, providing insights into the molecular mechanisms underlying channel dysfunction in migraine pathology. Statement of significanceThis study elucidates the biophysical properties of the Kv6.4 L360P mutation, providing insights into its role in Kv2.1 6.4 channel function. Given the high conservation of the leucine residue in the Kv channel family and its association with migraine, our findings have significant implications for understanding the molecular basis of migraine pathophysiology. By analyzing channels with a fixed 2:2 stoichiometry, we highlight the impact of the L360P mutation on channel gating and inactivation. This research advances the knowledge of the silent Kv6.4 channel mechanism and its role in pathophysiology.

biophysics↗

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

Pathways of inter- and intrasubunit allosteric signaling in the C-linker disk and cyclic nucleotide-binding domain of HCN2 channels

Opening of hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels is controlled by membrane hyperpolarization and binding of cyclic nucleotides to the tetrameric cyclic nucleotide-binding domain (CNBD), attached to the C-linker disk (CL). Confocal patch-clamp fluorometry revealed a pronounced cooperativity of ligand binding among protomers. However, by which pathways allosteric signal transmission occurs remained elusive. Here, we investigate how changes in the structural dynamics of the CL- CNBD of mouse HCN2 upon cAMP binding relate to inter- and intrasubunit signal transmission. Applying a rigidity theory-based approach, we identify two intersubunit and one intrasubunit pathways that differ in allosteric coupling strength between cAMP binding sites or towards the CL. These predictions agree with results from electrophysiological and patch-clamp fluorometry experiments. Our results map out distinct routes within the CL-CNBD that modulate different cAMP binding responses in HCN2 channels. They signify that functionally relevant submodules may exist within and across structurally discernable subunits in HCN channels.

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↗