Biomedical subjects

Martin Biel

Publications and source records attributed to Martin Biel.

6 recordsLinked to original sources

PubMed2026

Creating bottom-up RNA transfer vehicles from synthetic protein assemblies.

Evolution guides biological systems to populate ecological niches, with viruses among the most successful examples of this principle. Viruses evolved over billions of years to efficiently transfer genetic information. Although viruses are highly diverse, most have converged towards remarkable similarity in the size and shape of their capsids1,2. By contrast, generative models for protein design enable the creation of protein architectures that are absent from nature3-5. Here we investigate whether protein assemblies designed by artificial intelligence can be functionalized to construct nucleic acid transport vehicles that are independent of evolutionary trajectories. By combining natural protein domains with synthetic protein assemblies, we create more than 100 bottom-up RNA transfer vehicles with unique sizes and shapes. These vehicles surpass the RNA transfer efficiency of widely used delivery vehicles by several orders of magnitude. In addition, we demonstrate that their tropism can be programmed by incorporation of computationally designed peptide binders and use them to deliver therapeutically relevant cargo RNAs into a wide range of cellular models. We show the in vivo biodistribution of one of these vehicles in a mouse at near-single-cell resolution, confirm its safety, and use it to perform a gene-editing treatment strategy for Duchenne muscular dystrophy in patient-derived cells and a pig. Our work demonstrates how proteins created by generative artificial intelligence can be harnessed for the rational engineering of RNA transport systems with the desired properties by overcoming the limitations of natural protein diversity.

Journal Article↗

PubMed2003

Role of subunit heteromerization and N-linked glycosylation in the formation of functional hyperpolarization-activated cyclic nucleotide-gated channels.

The coassembly of homologous subunits to heteromeric complexes serves as an important mechanism in generating ion channel diversity. Here, we have studied heteromerization in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family. Using a combination of fluorescence confocal microscopy, coimmunoprecipitation, and electrophysiology we found that upon coexpression in HEK293 cells almost all dimeric combinations of HCN channel subunits give rise to the formation of stable channel complexes in the plasma membrane. We also identified HCN1/HCN2 heteromers in mouse brain indicating that heteromeric channels exist in vivo. Surprisingly, HCN2 and HCN3 did not coassemble to heteromeric channels. This finding indicates that heteromerization requires specific structural determinants that are not present in all HCN channel combinations. Using N-glycosidase F we show that native as well as recombinant HCN channels are glycosylated resulting in a 10-20-kDa shift in the molecular weight. Tunicamycin, an inhibitor of N-linked glycosylation, blocked surface membrane expression of HCN2. Similarly, a mutant HCN2 channel in which the putative N-glycosylation site in the loop between S5 and the pore helix was replaced by glutamine (HCN2N380Q) was not inserted into the plasma membrane and did not yield detectable whole-cell currents. These results indicate that N-linked glycosylation is required for cell surface trafficking of HCN channels. Cotransfection of HCN2N380Q with HCN4, but not with HCN3, rescued cell surface expression of HCN2N380Q. Immunoprecipitation revealed that this rescue was due to the formation of a HCN2N380Q/HCN4 heteromeric channel. Taken together our results indicate that subunit heteromerization and glycosylation are important determinants of the formation of native HCN channels.

Animals↗

PubMed2003

Molecular basis for the different activation kinetics of the pacemaker channels HCN2 and HCN4.

The pacemaker channels HCN2 and HCN4 have been identified in cardiac sino-atrial node cells. These channels differ considerably in several kinetic properties including the activation time constant (tau act), which is fast for HCN2 (144 ms at -140 mV) and slow for HCN4 (461 ms at -140 mV). Here, by analyzing HCN2/4 chimeras and mutants we identified single amino acid residues in transmembrane segments 1 and 2 and the connecting loop between S1 and S2 that are major determinants of this difference. Replacement of leucine 272 in S1 of HCN4 by the corresponding phenylalanine present in HCN2 decreased tau act of HCN4 to 149 ms. Conversely, activation of the fast channel HCN2 was decreased 3-fold upon the corresponding mutation of F221L in the S1 segment. Mutation of N291T and T293A in the linker between S1 and S2 of HCN4 shifted tau act to 275 ms. While residues 272, 291, and 293 of HCN4 affected the activation speed at basal conditions they had no obvious influence on the cAMP-dependent acceleration of activation kinetics. In contrast, mutation of I308M in S2 of HCN4 abolished the cAMP-dependent decrease in tau act. Surprisingly, this mutation also prevented the acceleration of channel activation observed after deletion of the C-terminal cAMP binding site. Taken together our results indicate that the speed of activation of the HCN4 channel is determined by structural elements present in the S1, S1-S2 linker, and the S2 segment.

Amino Acid Sequence↗

PubMed2003

Dominant-negative suppression of HCN channels markedly reduces the native pacemaker current I(f) and undermines spontaneous beating of neonatal cardiomyocytes.

BACKGROUND: The pacemaker current I(f) contributes to spontaneous diastolic depolarization of cardiac autonomic cells. In heterologous expression, HCN channels exhibit a hyperpolarization-activated inward current similar to I(f). However, the links between HCN genes and native I(f) are largely inferential, and it remains unknown whether I(f) is essential for cardiac pacing. METHODS AND RESULTS: To clarify this situation, we generated a GYG(402-404)AYA pore mutation of HCN2, which rendered the channel nonfunctional and suppressed wild-type HCN2 in a dominant-negative manner in Chinese hamster ovary cells. In addition, HCN2-AYA suppressed I(HCN4) in a dominant-negative manner when coexpressed with wild-type HCN4, indicating that the 2 isoforms HCN2 and HCN4 are able to coassemble to form heteromultimeric complexes. Given that HCN2 and HCN4 are the dominant HCN mRNA transcripts in neonatal rat ventricle, we expressed HCN2-AYA in neonatal cardiocytes using adenoviral gene transfer to test the effect of HCN suppression on native I(f). I(f) density was indeed reduced markedly, from 7.8+/-1.6 pA/pF (n=13) in control cells to 0.3+/-0.2 pA/pF (n=11) in HCN2-AYA-infected cells when measured at -130 mV (P<0.001). To probe the effect of HCN on cardiac pacing, we infected spontaneously beating neonatal monolayers with adenoviral vectors expressing wild-type and mutant HCN channels. Infection with HCN2 and HCN4 accelerated the beating rate significantly, to 230.5+/-8.6 bpm (n=12) and 223.5+/-12.3 bpm (n=10), respectively, compared with control cultures (83.4+/-4.5 bpm, n=13, P<0.001). Conversely, HCN2-AYA completely undermined spontaneous pacing of neonatal cardiocytes. CONCLUSIONS: HCN channels are the major molecular component of native I(f) and are critical for spontaneous beating of neonatal cardiomyocytes.

Adenoviridae↗

PubMed2003

Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2.

Hyperpolarization-activated cation (HCN) channels are believed to be involved in the generation of cardiac pacemaker depolarizations as well as in the control of neuronal excitability and plasticity. The contributions of the four individual HCN channel isoforms (HCN1-4) to these diverse functions are not known. Here we show that HCN2-deficient mice exhibit spontaneous absence seizures. The thalamocortical relay neurons of these mice displayed a near complete loss of the HCN current, resulting in a pronounced hyperpolarizing shift of the resting membrane potential, an altered response to depolarizing inputs and an increased susceptibility for oscillations. HCN2-null mice also displayed cardiac sinus dysrhythmia, a reduction of the sinoatrial HCN current and a shift of the maximum diastolic potential to hyperpolarized values. Mice with cardiomyocyte- specific deletion of HCN2 displayed the same dysrhythmia as mice lacking HCN2 globally, indicating that the dysrhythmia is indeed caused by sinoatrial dysfunction. Our results define the physiological role of the HCN2 subunit as a major determinant of membrane resting potential that is required for regular cardiac and neuronal rhythmicity.

Animals↗

PubMed2002

Cardiac HCN channels: structure, function, and modulation.

The hyperpolarization-activated cation current (termed I(f), I(h), or I(q)) plays a key role in the initiation and modulation of cardiac and neuronal pacemaker depolarizations. Recently, the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of ion channel subunits has been identified by molecular cloning. When heterologously expressed, each of the four HCN subunits (HCN1-4) generates channels with the principal properties of native I(f), indicating that HCN channels are the molecular correlate of this current. This review describes the molecular and functional diversity of the HCN channel family. The structural determinants of channel activation, modulation, and ion permeation are discussed. The expression pattern of HCN channels in different heart regions is reviewed. Finally, the relationships between biophysical properties of cloned HCN channel types and native cardiac I(f) are explored.

Amino Acid Sequence↗