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- Biophysics (3)
- Algal viruses (2)
- Codon Usage (2)
- Kcv (2)
- Molecular Physiology (2)
- TMEM175 (2)
- Viral K+ channel (2)
- effect of synonymous codon exchange (2)
- membrane protein sorting (2)
- rate constants (2)
BACKGROUND
Metabolic control and dietary management of patients with phenylketonuria (PKU) are based on single blood samples obtained at variable intervals. Sampling conditions are often not well-specified and intermittent variation of phenylalanine concentrations between two measurements remains unknown. We determined phenylalanine and tyrosine concentrations in blood over 24 hours. Additionally, the impact of food intake and physical exercise on phenylalanine and tyrosine concentrations was examined. Subcutaneous microdialysis was evaluated as a tool for monitoring phenylalanine and tyrosine concentrations in PKU patients.
METHODS
Phenylalanine and tyrosine concentrations of eight adult patients with PKU were determined at 60 minute intervals in serum, dried blood and subcutaneous microdialysate and additionally every 30 minutes postprandially in subcutaneous microdialysate. During the study period of 24 hours individually tailored meals with defined phenylalanine and tyrosine contents were served at fixed times and 20 min bicycle-ergometry was performed.
RESULTS
Serum phenylalanine concentrations showed only minor variations while tyrosine concentrations varied significantly more over the 24-hour period. Food intake within the patients' individual diet had no consistent effect on the mean phenylalanine concentration but the tyrosine concentration increased up to 300% individually. Mean phenylalanine concentration remained stable after short-term bicycle-exercise whereas mean tyrosine concentration declined significantly. Phenylalanine and tyrosine concentrations in dried blood were significantly lower than serum concentrations. No close correlation has been found between serum and microdialysis fluid for phenylalanine and tyrosine concentrations.
CONCLUSIONS
Slight diurnal variation of phenylalanine concentrations in serum implicates that a single blood sample does reliably reflect the metabolic control in this group of adult patients. Phenylalanine concentrations determined by subcutaneous microdialysis do not correlate with the patients' phenylalanine concentrations in serum/blood.
Some algal viruses have coding sequences for proteins with structural and functional characteristics of pore modules of complex K+ channels. Here we exploit the structural diversity among these channel orthologs to discover new basic principles of structure/function correlates in K+ channels. The analysis of three similar K+ channels with ≤ 86 amino acids (AA) shows that one channel (Kmpv1) generates an ohmic conductance in HEK293 cells while the other two (KmpvSP1, KmpvPL1) exhibit typical features of canonical Kir channels. Like Kir channels, the rectification of the viral channels is a function of the K+ driving force. Reconstitution of KmpvSP1 and KmpvPL1 in planar lipid bilayers showed rapid channel fluctuations only at voltages negative of the K+ reversal voltage. This rectification was maintained in KCl buffer with 1 mM EDTA, which excludes blocking cations as the source of rectification. This means that rectification of the viral channels must be an inherent property of the channel. The structural basis for rectification was investigated by a chimera between rectifying and non-rectifying channels as well as point mutations making the rectifier similar to the ohmic conducting channel. The results of these experiments exclude the pore with pore helix and selectivity filter as playing a role in rectification. The insensitivity of the rectifier to point mutations suggests that tertiary or quaternary structural interactions between the transmembrane domains are responsible for this type of gating.
Chloroviruses are large, plaque-forming, dsDNA viruses that infect chlorella-like green algae that live in a symbiotic relationship with protists. Chloroviruses have genomes from 290 to 370 kb, and they encode as many as 400 proteins. One interesting feature of chloroviruses is that they encode a potassium ion (K+) channel protein named Kcv. The Kcv protein encoded by SAG chlorovirus ATCV-1 is one of the smallest known functional K+ channel proteins consisting of 82 amino acids. The KcvATCV-1 protein has similarities to the family of two transmembrane domain K+ channel proteins; it consists of two transmembrane α-helixes with a pore region in the middle, making it an ideal model for studying K+ channels. To assess their genetic diversity, kcv genes were sequenced from 103 geographically distinct SAG chlorovirus isolates. Of the 103 kcv genes, there were 42 unique DNA sequences that translated into 26 new Kcv channels. The new predicted Kcv proteins differed from KcvATCV-1 by 1 to 55 amino acids. The most conserved region of the Kcv protein was the filter, the turret and the pore helix were fairly well conserved, and the outer and the inner transmembrane domains of the protein were the most variable. Two of the new predicted channels were shown to be functional K+ channels.
Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K+ channels, KcvS and KcvNTS. These small viral encoded K+ channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K+ channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in KcvNTS and 40% in KcvS. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in KcvS. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels.
Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers
(2018)
Recent studies have shown that membrane proteins can be efficiently synthesized in vitro before spontaneously inserting into soluble nanoscale lipid bilayers called nanodiscs (NDs). In this paper, we present experimental details that allow a combination of in vitro translation of ion channels into commercially available NDs followed by their direct reconstitution from these nanobilayers into standard bilayer setups for electrophysiological characterization. We present data showing that two model K+ channels, Kcv and KcsA, as well as a recently discovered dual-topology F- channel, Fluc, can be reliably reconstituted from different types of NDs into bilayers without contamination from the in vitro translation cocktail. The functional properties of Kcv and KcsA were characterized electrophysiologically and exhibited sensitivity to the lipid composition of the target DPhPC bilayer, suggesting that the channel proteins were fully exposed to the target membrane and were no longer surrounded by the lipid/protein scaffold. The single-channel properties of the three tested channels are compatible with studies from recordings of the same proteins in other expression systems. Altogether, the data show that synthesis of ion channels into NDs and their subsequent reconstitution into conventional bilayers provide a fast and reliable method for functional analysis of ion channels.
Plants acquire potassium (K+) ions for cell growth and movement via regulated diffusion through K+ channels. Here, we present crystallographic and functional data showing that the K+ inward rectifier KAT1 (K+Arabidopsis thaliana 1) channel is regulated by 14-3-3 proteins and further modulated by the phytotoxin fusicoccin, in analogy to the H+-ATPase. We identified a 14-3-3 mode III binding site at the very C terminus of KAT1 and cocrystallized it with tobacco (Nicotiana tabacum) 14-3-3 proteins to describe the protein complex at atomic detail. Validation of this interaction by electrophysiology shows that 14-3-3 binding augments KAT1 conductance by increasing the maximal current and by positively shifting the voltage dependency of gating. Fusicoccin potentiates the 14-3-3 effect on KAT1 activity by stabilizing their interaction. Crystal structure of the ternary complex reveals a noncanonical binding site for the toxin that adopts a novel conformation. The structural insights underscore the adaptability of fusicoccin, predicting more potential targets than so far anticipated. The data further advocate a common mechanism of regulation of the proton pump and a potassium channel, two essential elements in K+ uptake in plant cells.
Lipid bilayers provide many benefits for ion channel recordings, such as control of membrane composition and transport molecules. However, they suffer from high membrane capacitance limiting the bandwidth and impeding analysis of fast gating. This can be overcome by fitting the deviations of the open-channel noise from the baseline noise by extended beta distributions. We demonstrate this analysis step-by-step by applying it to the example of viral K+ channels (Kcv), from the choice of the gating model through the fitting process, validation of the results, and what kinds of results can be obtained. These voltage sensor-less channels show profoundly voltage-dependent gating with dwell times in the closed state of about 50 μs. Mutations assign it to the selectivity filter.
Phycodnaviruses are dsDNA viruses, which infect algae. Their large genomes encode many gene products, like small K(+) channels, with homologs in prokaryotes and eukaryotes. Screening for K(+) channels revealed their abundance in viruses from fresh-water habitats. Recent sequencing of viruses from marine algae or from salt water in Antarctica revealed sequences with the predicted characteristics of K(+) channels but with some unexpected features. Two genes encode either 78 or 79 amino acid proteins, which are the smallest known K(+) channels. Also of interest is an unusual sequence in the canonical α-helixes in K(+) channels. Structural prediction algorithms indicate that the new channels have the conserved α-helix folds but the algorithms failed to identify the expected transmembrane domains flanking the K(+) channel pores. In spite of these unexpected properties electophysiological studies confirmed that the new proteins are functional K(+) channels.
Due to the redundancy of the genetic code most amino acids are encoded by multiple synonymous codons. It has been proposed that a biased frequency of synonymous codons can affect the function of proteins by modulating distinct steps in transcription, translation and folding. Here, we use two similar prototype K+ channels as model systems to examine whether codon choice has an impact on protein sorting. By monitoring transient expression of GFP-tagged channels in mammalian cells, we find that one of the two channels is sorted in a codon and cell cycle-dependent manner either to mitochondria or the secretory pathway. The data establish that a gene with either rare or frequent codons serves, together with a cell-state-dependent decoding mechanism, as a secondary code for sorting intracellular membrane proteins.
The calculation of flux equations or current-voltage relationships in reaction kinetic models with a high number of states can be very cumbersome. Here, a recipe based on an arrow scheme is presented, which yields a straightforward access to the minimum form of the flux equations and the occupation probability of the involved states in cyclic and linear reaction schemes. This is extremely simple for cyclic schemes without branches. If branches are involved, the effort of setting up the equations is a little bit higher. However, also here a straightforward recipe making use of so-called reserve factors is provided for implementing the branches into the cyclic scheme, thus enabling also a simple treatment of such cases.
Voltage-dependent anion-selective channel (VDAC) is one of the main proteins of the outer mitochondrial membrane of all eukaryotes, where it forms aqueous, voltage-sensitive, and ion-selective channels. Its electrophysiological properties have been thoroughly analyzed with the planar lipid bilayer technique. To date, however, available results are based on isolations of VDACs from tissue or from recombinant VDACs produced in bacterial systems. It is well known that the cytosolic overexpression of highly hydrophobic membrane proteins often results in the formation of inclusion bodies containing insoluble aggregates. Purification of properly folded proteins and restoration of their full biological activity requires several procedures that considerably lengthen experimental times. To overcome these restraints, we propose a one-step reaction that combines in vitro cell-free protein expression with nanodisc technology to obtain human VDAC isoforms directly integrated in a native-like lipid bilayer. Reconstitution assays into artificial membranes confirm the reliability of this new methodological approach and provide results comparable to those of VDACs prepared with traditional protein isolation and reconstitution protocols. The use of membrane-mimicking nanodisc systems represents a breakthrough in VDAC electrophysiology and may be adopted to further structural studies.
Transmembrane protein 175 (TMEM175) is an endolysosomal cation channel, which has attracted much attention recently from academics and the pharmaceutical industry alike since human mutations in TMEM175 were found to be associated with the development of Parkinson's disease (PD). Thus, gain-of-function mutations were identified, which reduce and loss-of-function mutations, which increase the risk of developing PD. After having been characterized as an endolysosomal potassium channel initially, soon after TMEM175 was claimed to act as a proton channel. In fact, recent evidence suggests that depending on the conditions, TMEM175 can act as either a potassium or proton channel, without acting as an antiporter or exchanger. A recent work has now identified amino acid H57 to be directly involved in gating, increasing proton conductance of the channel while leaving the potassium conductance unaffected. We review here the current knowledge of TMEM175 function, pharmacology, physiology, and pathophysiology. We discuss the potential of this ion channel as a novel drug target for the treatment of neurodegenerative diseases such as PD, and we discuss the discovery of H57 as proton sensor.
The lysosomal cation channel TMEM175 is crucial for maintaining lysosomal function and pH homeostasis, and its aberrant function is linked to Parkinson’s disease (PD). While TMEM175 activity was first interpreted in the context of its potassium (K+) selective conductance, subsequent studies revealed also a substantial permeability to protons (H+). Here we dissect the complex changes in TMEM175 conductance and current reversal voltages in response to pH jumps on the luminal side of the channel protein. In whole-cell patch clamp experiments with plasma membrane redistributed TMEM175 we show that a pH jump from symmetrical pH 7.4 to pH 4.7 on the luminal side triggers a continuous rise in inward and outward current, concomitant with a transient positive excursion of the reversal voltage (Erev). The peak Erev shift remains almost 100 mV below the estimated equilibrium voltage for protons and shows little sensitivity to the K+ gradient. The data are consistent with a scenario in which a TMEM175 mediated proton flux elicits a fast collapse of the pH gradient. In MD simulations we identify the luminal H57 as titratable partner for the formation of intra- and inter-subunit salt bridges with D279 and E282 for stabilizing the channel open state. This presumed gating function is confirmed by mutational studies and lysosomal patch-clamp experiments in which a H57Y mutant exhibits a reduced pH dependency of activation. Our findings contribute to a better comprehension of TMEM175’s complex electrophysiological properties and foster understanding of TMEM175 as a pharmacological target for neurodegenerative disease therapy.
The heart tissue is a potential target of various noxae contributing to the onset of cardiovascular diseases. However, underlying pathophysiological mechanisms are largely unknown. Human stem cell-derived models are promising, but a major concern is cell immaturity when estimating risks for adults. In this study, 3D aggregates of human embryonic stem cell-derived cardiomyocytes were cultivated for 300 days and characterized regarding degree of maturity, structure, and cell composition. Furthermore, effects of ionizing radiation (X-rays, 0.1-2 Gy) on matured aggregates were investigated, representing one of the noxae that are challenging to assess. Video-based functional analyses were correlated to changes in the proteome after irradiation. Cardiomyocytes reached maximum maturity after 100 days in cultivation, judged by α-actinin lengths, and displayed typical multinucleation and branching. At this time, aggregates contained all major cardiac cell types, proven by the patch-clamp technique. Matured and X-ray-irradiated aggregates revealed a subtle increase in beat rates and a more arrhythmic sequence of cellular depolarisation and repolarisation compared to non-irradiated sham controls. The proteome analysis provides first insights into signaling mechanisms contributing to cardiotoxicity. Here, we propose an in vitro model suitable to screen various noxae to target adult cardiotoxicity by preserving all the benefits of a 3D tissue culture.
Hyperpolarization-activated cyclic-nucleotide gated (HCN) channels are important for timing biological processes like heartbeat and neuronal firing. Their weak cation selectivity is determined by a filter domain with only two binding sites for K+ and one for Na+. The latter acts as a weak blocker, which is released in combination with a dynamic widening of the filter by K+ ions, giving rise to a mixed K+/Na+ current. Here, we apply molecular dynamics simulations to systematically investigate the interactions of five alkali metal cations with the filter of the open HCN4 pore. Simulations recapitulate experimental data like a low Li+ permeability, considerable Rb+ conductance, a block by Cs+ as well as a punch through of Cs+ ions at high negative voltages. Differential binding of the cation species in specific filter sites is associated with structural adaptations of filter residues. This gives rise to ion coordination by a cation-characteristic number of oxygen atoms from the filter backbone and solvent. This ion/protein interplay prevents Li+, but not Na+, from entry into and further passage through the filter. The site equivalent to S3 in K+ channels emerges as a preferential binding and presumably blocking site for Cs+. Collectively, the data suggest that the weak cation selectivity of HCN channels and their block by Cs+ are determined by restrained cation-generated rearrangements of flexible filter residues.
Mutation in pore-helix modulates interplay between filter gate and Ba2+ block in a Kcv channel pore
(2024)
The selectivity filter of K+ channels catalyzes a rapid and highly selective transport of K+ while serving as a gate. To understand the control of this filter gate, we use the pore-only K+ channel KcvNTS in which gating is exclusively determined by the activity of the filter gate. It has been previously shown that a mutation at the C-terminus of the pore-helix (S42T) increases K+ permeability and introduces distinct voltage-dependent and K+-sensitive channel closures at depolarizing voltages. Here, we report that the latter are not generated by intrinsic conformational changes of the filter gate but by a voltage-dependent block caused by nanomolar trace contaminations of Ba2+ in the KCl solution. Channel closures can be alleviated by extreme positive voltages and they can be completely abolished by the high-affinity Ba2+ chelator 18C6TA. By contrast, the same channel closures can be augmented by adding Ba2+ at submicromolar concentrations to the cytosolic buffer. These data suggest that a conservative exchange of Ser for Thr in a crucial position of the filter gate increases the affinity of the filter for Ba2+ by >200-fold at positive voltages. While Ba2+ ions apparently remain only for a short time in the filter-binding sites of the WT channel before passing the pore, they remain much longer in the mutant channel. Our findings suggest that the dwell times of permeating and blocking ions in the filter-binding sites are tightly controlled by interactions between the pore-helix and the selectivity filter.
When the K+ channel-like protein Kesv from Ectocarpus siliculosus virus 1 is heterologously expressed in mammalian cells, it is sorted to the mitochondria. This targeting can be redirected to the endoplasmic reticulum (ER) by altering the codon usage in distinct regions of the gene or by inserting a triplet of hydrophobic amino acids (AAs) into the protein's C-terminal transmembrane domain (ct-TMD). Systematic variations in the flavor of the inserted AAs and/or its codon usage show that a positive charge in the inserted AA triplet alone serves as strong signal for mitochondria sorting. In cases of neutral AA triplets, mitochondria sorting are favored by a combination of hydrophilic AAs and rarely used codons; sorting to the ER exhibits the inverse dependency. This propensity for ER sorting is particularly high when a common codon follows a rarer one in the AA triplet; mitochondria sorting in contrast is supported by codon uniformity. Since parameters like positive charge, hydrophobic AAs, and common codons are known to facilitate elongation of nascent proteins in the ribosome the data suggest a mechanism in which local changes in elongation velocity and co-translational folding in the ct-TMD influence intracellular protein sorting.
Distinct lipid bilayer compositions have general and protein-specific effects on K+ channel function
(2021)
It has become increasingly apparent that the lipid composition of cell membranes affects the function of transmembrane proteins such as ion channels. Here, we leverage the structural and functional diversity of small viral K+ channels to systematically examine the impact of bilayer composition on the pore module of single K+ channels. In vitro-synthesized channels were reconstituted into phosphatidylcholine bilayers ± cholesterol or anionic phospholipids (aPLs). Single-channel recordings revealed that a saturating concentration of 30% cholesterol had only minor and protein-specific effects on unitary conductance and gating. This indicates that channels have effective strategies for avoiding structural impacts of hydrophobic mismatches between proteins and the surrounding bilayer. In all seven channels tested, aPLs augmented the unitary conductance, suggesting that this is a general effect of negatively charged phospholipids on channel function. For one channel, we determined an effective half-maximal concentration of 15% phosphatidylserine, a value within the physiological range of aPL concentrations. The different sensitivity of two channel proteins to aPLs could be explained by the presence/absence of cationic amino acids at the interface between the lipid headgroups and the transmembrane domains. aPLs also affected gating in some channels, indicating that conductance and gating are uncoupled phenomena and that the impact of aPLs on gating is protein specific. In two channels, the latter can be explained by the altered orientation of the pore-lining transmembrane helix that prevents flipping of a phenylalanine side chain into the ion permeation pathway for long channel closings. Experiments with asymmetrical bilayers showed that this effect is leaflet specific and most effective in the inner leaflet, in which aPLs are normally present in plasma membranes. The data underscore a general positive effect of aPLs on the conductance of K+ channels and a potential interaction of their negative headgroup with cationic amino acids in their vicinity.
Potassium ion (K+) channels have been observed in diverse viruses that infect eukaryotic marine and freshwater algae. However, experimental evidence for functional K+ channels among these alga-infecting viruses has thus far been restricted to members of the family Phycodnaviridae, which are large, double-stranded DNA viruses within the phylum Nucleocytoviricota. Recent sequencing projects revealed that alga-infecting members of Mimiviridae, another family within this phylum, may also contain genes encoding K+ channels. Here we examine the structural features and the functional properties of putative K+ channels from four cultivated members of Mimiviridae. While all four proteins contain variations of the conserved selectivity filter sequence of K+ channels, structural prediction algorithms suggest that only two of them have the required number and position of two transmembrane domains that are present in all K+ channels. After in vitro translation and reconstitution of the four proteins in planar lipid bilayers, we confirmed that one of them, a 79 amino acid protein from the virus Tetraselmis virus 1 (TetV-1), forms a functional ion channel with a distinct selectivity for K+ over Na+ and a sensitivity to Ba2+. Thus, virus-encoded K+ channels are not limited to Phycodnaviridae but also occur in the members of Mimiviridae. The large sequence diversity among the viral K+ channels implies multiple events of lateral gene transfer.
Yeast complementation assays provide limited informationon functional features of K+ channels
(2025)
We investigate to what extent yeast complementation assays, which in principle can provide large amounts of training data for machine learning models, yield quantitative correlations between growth rescue and single channel recordings. If this were the case, yeast complementation results could be used as surrogate data for machine learning-based channel design. Therefore, we mutated position L94 at the cavity entry of the model K+ channel KcvPBCV1 to all proteinogenic amino acids. The function of the WT channel and its mutants was investigated by reconstituting them in planar lipid bilayers and by their ability to rescue the growth of a yeast strain deficient in K+ uptake. The single channel data show a distinct effect of mutations in this critical position on unitary conductance and open probability, with no apparent causal relationship between the two functional parameters. We also found that even conservative amino acid replacements can alter the unitary conductance and/or open probability and that most functional changes show no systematic relationship with the physicochemical nature of the amino acids. This emphasizes that the functional influence of an amino acid on channel function cannot be reduced to a single chemical property. Mutual comparison of single channel data and yeast complementation results exhibit only a partial correlation between their electrical parameters and their potency of rescuing growth. Hence complementation data alone are not sufficient for enabling functional channel design; they need to be complemented by additional parameters like the number of channels in the plasma membrane.
