Biology subjects

Karbat, I.

Publications and source records attributed to Karbat, I..

5 recordsLinked to original sources

bioRxiv2023

Comment on 'Connexins evolved after early chordates lost innexin diversity'

Gap junctional (GJ) intercellular channels are an important way of intercellular communication. Currently, two unrelated families of GJ proteins, innexins/pannexins, and connexins, are known, either or both of which are present in most multicellular animals. A striking exception is the echinoderms which have functional GJs but until recently were believed to be lacking both innexins and connexins, which suggests the presence of the third, yet the unknown family of GJ proteins. In the recent work Welzel and Schuster (Welzel and Schuster, 2022) have reported several putative innexins and one connexin from echinoderms, therefore undermining such a hypothesis. Here we provide evidence showing that all reported connexin and innexin sequences from echinoderms are cross-species contaminations, indicating that a search for a third GJ protein family is still a subject of immediate scientific research interest.

evolutionary biology↗

bioRxiv2022

One-shot design elevates functional expression levels of a voltage-gated potassium channel

Membrane proteins play critical physiological roles as receptors, channels, pumps, and transporters. Despite their importance, however, low expression levels often hamper the experimental characterization of membrane proteins. We present an automated and web-accessible design algorithm called mPROSS (https://mPROSS.weizmann.ac.il), which uses phylogenetic analysis and an atomistic potential, including an empirical lipophilicity scale, to improve native-state energy. As a stringent test, we apply mPROSS to the Kv1.2-Kv2.1 paddle chimera voltage-gated potassium channel. Four designs, encoding 9-26 mutations relative to the parental channel, were functional and maintained potassium-selective permeation and voltage dependence in Xenopus oocytes with up to 14-fold increase in whole-cell current densities. Additionally, single-channel recordings reveal no significant change in the channel-opening probability nor in unitary conductance, indicating that functional expression levels increase without impacting the activity profile of individual channels. Our results suggest that the expression levels of other dynamical channels and receptors may be enhanced through one-shot design calculations. Significance statementHeterologous expression levels of membrane proteins are often low, limiting research and applications. We combine homologous-sequence analysis with Rosetta atomistic calculations to enable one-shot design of dozens of mutations that improve native-state energy. Applied to a voltage-gated potassium channel, designs exhibited up to 14-fold improved functional expression levels in oocytes with almost no change in the single-channel activity profile. This design approach may accelerate research of many challenging membrane proteins, including receptors, channels, and transporters.

biochemistry↗

bioRxiv2022

Store-operated Ca2+ entry regulatory factor (SARAF) alters murine metabolic state in an age-dependent manner via hypothalamic pathways.

Store-operated Ca2+ entry (SOCE) is a vital process aimed at refilling cellular internal Ca2+ stores, and a primary cellular-signaling driver of transcription factors entry to the nucleus. SARAF (SOCE associated regulatory factor)/TMEM66 is an endoplasmic reticulum (ER) resident transmembrane protein that promotes SOCE inactivation and prevents Ca2+ overfilling of the cell. Here we demonstrate that mice deficient in SARAF develop age-dependent sarcopenic obesity with decreased energy expenditure, lean mass and locomotion without affecting food consumption. Moreover, SARAF ablation reduces hippocampal proliferation, modulates the activity of the hypothalamus-pituitary-adrenal (HPA) axis, and mediates changes in anxiety-related behaviors. Interestingly, selective SARAF ablation in the paraventricular nucleus (PVN) of the hypothalamus protects from old age-induced obesity and preserves locomotion, lean mass and energy expenditure, suggesting an opposing, site-specific role for SARAF. Lastly, SARAF ablation in hepatocytes leads to elevated SOCE, elevated vasopressin-induced Ca2+ oscillations, and an increased mitochondrial spare respiratory capacity, thus providing insights into the cellular mechanisms that may affect the global phenotypes. These effects may be mediated via the liver X receptor (LXR) and IL-1 signaling metabolic regulators explicitly altered in SARAF ablated cells. In short, our work supports both central and peripheral roles of SARAF in regulating metabolic, behavioral, and cellular responses. HighlightsO_LILoss/absence of SARAF facilitates age-dependent obesity with decreased metabolic rate, lean mass, and locomotion, without affecting food consumption. C_LIO_LILoss of SARAF leads to lipid droplet hypertrophy, BAT whitening and age-dependent hepatic steatosis. C_LIO_LIMice lacking SARAF expression in the PVN have an increased metabolic rate, decreased BAT whitening, and are protected from sarcopenic obesity. C_LIO_LISARAF ablation in hepatocytes increases SOCE, elevates Ca2+ oscillation in response to vasopressin, and increases the mitochondrias spare respiratory capacity. C_LIO_LILoss of SARAF leads to decreased hippocampal proliferation, sensitized HPA-axis and changes in anxiety-related behavior. C_LI

physiology↗

bioRxiv2020

A molecular lid mechanism for K+ channel blockers revealed by a cone peptide

Many venomous organisms carry in their arsenal short polypeptides that block K+ channels in a highly selective manner. These toxins may compete with the permeating ions directly via a “plug” mechanism or indirectly via a “pore-collapse” mechanism. An alternative “lid” mechanism was proposed but remained poorly defined. Here we study the block of the Drosophila Shaker channel by Conknunitzin-S1 and Conkunitzin-C3, two highly similar toxins derived from cone venom. Despite their similarity, the two peptides exhibited differences in their binding poses and in biophysical assays, implying discrete modes of action. We show that while Conknunitzin-S1 binds tightly to the channel turret and acts via a “pore-collapse” mechanism, Conkunitzin-C3 does not contact this region. Instead, Conk-C3 uses a non-conserved Arg to divert the permeant ions and trap them in off-axis cryptic sites above the SF, a mechanism we term a “molecular-lid”. Our study provides an atomic description of the “lid” K+ blocking mode and offers valuable insights for the design of therapeutics based on venom peptides.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2019

Pore-modulating toxins exploit inherent slow inactivation to block K+ channels

Voltage dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified to \"pore-blockers\" that physically plug the ion conduction pathway and \"gating modifiers\" that disrupt voltage sensor movements. A third group of toxins blocks K+ conduction by an unknown mechanism via binding to the channel turrets. Here we show that Cs1, a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+ channels as a novel pharmacological target and provides a rational framework for drug design.

pharmacology and toxicology↗