Biology subjects

De Gieter, S.

Publications and source records attributed to De Gieter, S..

3 recordsLinked to original sources

bioRxiv2025

Structural basis for gating inhibition by the cytoplasmic domain in HCN1 channels

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate rhythmic electrical activity in cardiac and neuronal tissues, with isoform-specific cAMP sensitivity remaining poorly understood. While HCN2 exhibits strong cAMP regulation, HCN1 shows minimal response. To investigate the structural basis of this divergence, we analyzed two engineered HCN1 variants using cryo-electron microscopy. One variant (HCN112) incorporates the C-linker and CNBD from HCN2 into the HCN1 backbone and exhibited enhanced cAMP sensitivity, with structural analysis revealing a compressed cytoplasmic domain arrangement that may facilitate regulatory interactions. In contrast, the truncated HCN1{Delta}C variant (lacking the cytoplasmic domain) displayed an intermediately open pore conformation, supporting auto-inhibitory regulation by the CNBD in wild-type channels. These structural insights elucidate how domain-specific interactions modulate cAMP-dependent gating and intrinsic auto-inhibition, resolving long-standing questions about mechanistic divergence among HCN isoforms. Our findings not only shed new light on the structural mechanisms underlying isoform-specific cAMP sensitivity but also have implications for the development of therapeutic strategies targeting HCN channels in neurological and cardiac disorders. SIGNIFICANCE STATEMENTHyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential regulators of rhythmic electrical activity in the heart and brain, and their dysfunction is linked to disorders such as epilepsy, depression, and cardiac arrhythmias. Although HCN channel isoforms display highly divergent responses to cAMP modulation, the structural basis for these differences has remained elusive. This study reveals, through cryo-EM structures, how domain-specific interactions within the cytoplasmic regions of HCN1 channels underlie their unique gating and auto-inhibition properties, as well as their muted cAMP sensitivity compared to HCN2. These insights resolve long-standing mechanistic questions about HCN channel regulation and pave the way for rational design of targeted therapies that selectively modulate HCN isoform activity in neurological and cardiac disease.

biochemistry↗

bioRxiv2025

MISO: Microfluidic protein isolation enables single particle cryo-EM structure determination from a single cell colony

Single particle cryo-EM enables reconstructing near-atomic or even atomic resolution 3D maps of proteins by visualizing thousands to a few million purified protein particles embedded in nanometer- thick vitreous ice. This corresponds to picograms of purified protein, which can potentially be isolated from a few thousand cells. Hence, cryo-EM holds the potential of one of the most sensitive analytical methods that deliver a high-resolution protein structure as a readout. In practice, more than a million times more starting biological material is required to prepare cryo-EM grids. To close the gap, we developed a micro isolation (MISO) method that combines microfluidics-based protein purification with cryo-EM grid preparation. We validated the method on soluble bacterial and eukaryotic membrane proteins. We showed that MISO enables protein structure determination starting from below one microgram of a target protein and going from cells to cryo-EM grids within a few hours. This scales down the purification by a factor of a few hundred to a few thousand and opens possibilities for the structural characterization of hitherto inaccessible proteins.

biophysics↗

bioRxiv2023

Sterol derivative binding to the orthosteric site causes conformational changes in an invertebrate Cys-loop receptor

Cys-loop receptors or pentameric ligand-gated ion channels are mediators of electrochemical signaling throughout the animal kingdom. Because of their critical function in neurotransmission and high potential as drug targets, Cys-loop receptors from humans and closely related organisms have been thoroughly investigated, whereas molecular mechanisms of neurotransmission in invertebrates are less understood. When compared with vertebrates, the invertebrate genomes underwent a drastic expansion in the number of the nACh-like genes associated with receptors of unknown function. Understanding this diversity contributes to better insight into the evolution and possible functional divergence of these receptors. In this work, we studied orphan receptor Alpo4 from an extreme thermophile worm Alvinella pompejana. Sequence analysis points towards its remote relation to characterized nACh receptors. We solved the first cryo-EM structure of a lophotrochozoan nACh-like receptor in which a CHAPS molecule is tightly bound to the orthosteric site. We show that the binding of CHAPS leads to extending of the loop C at the orthosteric site and a clockwise quaternary twist between extracellular and transmembrane domains. Both the ligand binding site and the channel pore reveal unique features. These include a conserved Trp residue in loop B of the ligand binding site which is flipped into an apparent self-liganded state in the apo structure. The ion pore of Alpo4 is tightly constricted by a ring of methionines near the extracellular entryway of the channel pore. Our data provide a structural basis for a functional understanding of Alpo4 and hints towards new strategies for designing specific channel modulators.

biochemistry↗