Ancestral acetylcholine receptor β-subunit forms homopentamers that prime before opening spontaneously

Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from two α-subunits, and one each of the β-, δ-, and ϵ- subunits. To form functional channels, the subunits must assemble with one another in a precise stoichiometry and arrangement. Despite being different, the four subunits share a common ancestor that is presumed to have formed homopentamers. The extent to which the properties of the modern-day receptor result from its subunit complexity is unknown. Here we show that a reconstructed ancestral muscle-type β-subunit can form homopentameric ion channels. These homopentamers open spontaneously and display single-channel hallmarks of muscle type acetylcholine receptor activity. Our findings demonstrate that signature features of muscle-type acetylcholine receptor function are independent of agonist, and do not necessitate the complex heteropentameric architecture of the modern-day receptor.

Transcriptional expressions of SCNs family correlates with overall survival in patients with breast cancer

Introduction: Voltage-gated sodium (Nav) channels encoded by SCNs are heteromeric protein complexes containing pore-forming α subunits together with non-pore-forming β subunits. Methods: To analyze the expression of SCNs in the samples of different types of breast cancer (BC) patients and the relationship between the expression of α and β subunits and the prognosis of in BC patients, the study investigated the roles of SCNs in the prognosis of BC using ONCOMINE, UALCAN, Kaplan-Meier Plotter, GEPIA, Metascape, LinkedOmics databases. The study analyzed significant changes of SCNs expression and prognosis in transcription level between BC and normal samples, and association of mRNA expression of distinct SCNs family members with prognosis in overall BC patients and HER2-positive/HER2-negative subgroups, respectively. Moreover, we predicted functions and pathways of the mutations in SCNs and their neighbor genes in BC patients by GO/KEGG and GSEA analysis. Results: The results showed that transcriptional and proteinic expressions of 9 SCNs were downregulated in patients with BC, including SCN1A~4A, 7A, 9A and SCN2B~4B. low expressions of 11 SCNs members were found to be significantly associated with poorer overall survival (OS) in BC patients (P＜0.01), including SCN2A, 3A, 5A, 7A, 9A~11A and SCN1B~4B. Moreover, prognostic value of mRNA expression of SCNs could only be seen in HER2-negative BC patients when we performed subgroup analysis. Conclusions: These results indicated that SCNs could be prognostic biomarkers for survivals of BC patients. Some medicines that regulate SCNs might provide new targets for BC treatment.

Oxygen-sensing mechanisms in development and tissue repair

ABSTRACT Under normoxia, hypoxia inducible factor (HIF) α subunits are hydroxylated by PHDs (prolyl hydroxylase domain proteins) and subsequently undergo polyubiquitylation and degradation. Normal embryogenesis occurs under hypoxia, which suppresses PHD activities and allows HIFα to stabilize and regulate development. In this Primer, we explain molecular mechanisms of the oxygen-sensing pathway, summarize HIF-regulated downstream events, discuss loss-of-function phenotypes primarily in mouse development, and highlight clinical relevance to angiogenesis and tissue repair.

Structure of the ATP synthase from Mycobacterium smegmatis provides targets for treating tuberculosis

The structure has been determined by electron cryomicroscopy of the adenosine triphosphate (ATP) synthase from Mycobacterium smegmatis. This analysis confirms features in a prior description of the structure of the enzyme, but it also describes other highly significant attributes not recognized before that are crucial for understanding the mechanism and regulation of the mycobacterial enzyme. First, we resolved not only the three main states in the catalytic cycle described before but also eight substates that portray structural and mechanistic changes occurring during a 360° catalytic cycle. Second, a mechanism of auto-inhibition of ATP hydrolysis involves not only the engagement of the C-terminal region of an α-subunit in a loop in the γ-subunit, as proposed before, but also a “fail-safe” mechanism involving the b′-subunit in the peripheral stalk that enhances engagement. A third unreported characteristic is that the fused bδ-subunit contains a duplicated domain in its N-terminal region where the two copies of the domain participate in similar modes of attachment of the two of three N-terminal regions of the α-subunits. The auto-inhibitory plus the associated “fail-safe” mechanisms and the modes of attachment of the α-subunits provide targets for development of innovative antitubercular drugs. The structure also provides support for an observation made in the bovine ATP synthase that the transmembrane proton-motive force that provides the energy to drive the rotary mechanism is delivered directly and tangentially to the rotor via a Grotthuss water chain in a polar L-shaped tunnel.

SUMOylation of the Kv4.2 Ternary Complex Increases Surface Expression and Current Amplitude by Reducing Internalization in HEK 293 Cells

Kv4 α-subunits exist as ternary complexes (TC) with potassium channel interacting proteins (KChIP) and dipeptidyl peptidase-like proteins (DPLP); multiple ancillary proteins also interact with the α-subunits throughout the channel’s lifetime. Dynamic regulation of Kv4.2 protein interactions adapts the transient potassium current, IA, mediated by Kv4 α-subunits. Small ubiquitin-like modifier (SUMO) is an 11 kD peptide post-translationally added to lysine (K) residues to regulate protein–protein interactions. We previously demonstrated that when expressed in human embryonic kidney (HEK) cells, Kv4.2 can be SUMOylated at two K residues, K437 and K579. SUMOylation at K437 increased surface expression of electrically silent channels while SUMOylation at K579 reduced IA maximal conductance (Gmax) without altering surface expression. KChIP and DPLP subunits are known to modify the pattern of Kv4.2 post-translational decorations and/or their effects. In this study, co-expressing Kv4.2 with KChIP2a and DPP10c altered the effects of enhanced Kv4.2 SUMOylation. First, the effect of enhanced SUMOylation was the same for a TC containing either the wild-type Kv4.2 or the mutant K437R Kv4.2, suggesting that either the experimental manipulation no longer enhanced K437 SUMOylation or K437 SUMOylation no longer influenced Kv4.2 surface expression. Second, instead of decreasing IA Gmax, enhanced SUMOylation at K579 now produced a significant ∼37–70% increase in IA maximum conductance (Gmax) and a significant ∼30–50% increase in Kv4.2g surface expression that was accompanied by a 65% reduction in TC internalization. Blocking clathrin-mediated endocytosis (CME) in HEK cells expressing the Kv4.2 TC mimicked and occluded the effect of SUMO on IA Gmax; however, the amount of Kv4.2 associated with the major adaptor for constitutive CME, adaptor protein 2 (AP2), was not SUMO dependent. Thus, SUMOylation reduced Kv4.2 internalization by acting downstream of Kv4.2 recruitment into clathrin-coated pits. In sum, the two major findings of this study are: SUMOylation of Kv4.2 at K579 regulates TC internalization most likely by promoting channel recycling. Additionally, there is a reciprocity between Kv4.2 SUMOylation and the Kv4.2 interactome such that SUMOylation regulates the interactome and the interactome influences the pattern and effect of SUMOylation.

Transcriptional Expressions of SCNs Family Correlates With Overall Survival in Patients With Breast Cancer

Background: Voltage-gated sodium (Nav) channels encoded by SCNs are heteromeric protein complexes containing pore-forming α subunits together with non-pore-forming β subunits. Ion channels play an important role in the regulation of many cellular processes during normal physiology, and increasingly recognized for their contribution to pathophysiology, including cancers. Numerous studies in the last years have reported the expression of SCNs in metastatic cells of many cancers and their upregulation have been evident in promoting migration, invasion and metastasis, whereas it remains unclear whether distinct SCNs family members play an important role in the development and progression of BC. Results: The study investigated the roles of SCNs in the prognosis of BC using ONCOMINE, UALCAN, Kaplan-Meier Plotter, GEPIA, Metascape, LinkedOmics databases. The results showed that transcriptional and proteinic expressions of 9 SCNs were downregulated in patients with BC, including SCN1A~4A, 7A, 9A and SCN2B~4B. low expressions of 11 SCNs members were found to be significantly associated with poorer overall survival (OS) in BC patients (P＜0.01), including SCN2A, 3A, 5A, 7A, 9A~11A and SCN1B~4B. Moreover, prognostic value of mRNA expression of SCNs could only be seen in HER2-negative BC patients when we performed subgroup analysis. Conclusions: These results indicated that SCNs could be prognostic biomarkers for survivals of BC patients. Some medicines that regulate SCNs might provide new targets for BC treatment.

A Chaperone-Like Role for EBI3 in Collaboration With Calnexin Under Inflammatory Conditions

The interleukin-6 (IL-6)/IL-12 family of cytokines plays critical roles in the induction and regulation of innate and adaptive immune responses. Among the various cytokines, only this family has the unique characteristic of being composed of two distinct subunits, α- and β-subunits, which form a heterodimer with subunits that occur in other cytokines as well. Recently, we found a novel intracellular role for one of the α-subunits, Epstein-Barr virus-induced gene 3 (EBI3), in promoting the proper folding of target proteins and augmenting its expression at the protein level by binding to its target protein and a well-characterized lectin chaperone, calnexin, presumably through enhancing chaperone activity. Because calnexin is ubiquitously and constitutively expressed but EBI3 expression is inducible, these results could open an avenue to establish a new paradigm in which EBI3 plays an important role in further increasing the expression of target molecules at the protein level in collaboration with calnexin under inflammatory conditions. This theory well accounts for the heterodimer formation of EBI3 with p28, and probably with p35 and p19 to produce IL-27, IL-35, and IL-39, respectively. In line with this concept, another β-subunit, p40, plays a critical role in the assembly-induced proper folding of p35 and p19 to produce IL-12 and IL-23, respectively. Thus, chaperone-like activities in proper folding and maturation, which allow the secretion of biologically active heterodimeric cytokines, have recently been highlighted. This review summarizes the current understanding of chaperone-like activities of EBI3 to form heterodimers and other associations together with their possible biological implications.

Inwardly rectifying potassium channels (KIR) in GtoPdb v.2021.3

The 2TM domain family of K channels are also known as the inward-rectifier K channel family. This family includes the strong inward-rectifier K channels (Kir2.x) that are constitutively active, the G-protein-activated inward-rectifier K channels (Kir3.x) and the ATP-sensitive K channels (Kir6.x, which combine with sulphonylurea receptors (SUR1-3)). The pore-forming α subunits form tetramers, and heteromeric channels may be formed within subfamilies (e.g. Kir3.2 with Kir3.3).

Molecular determinants of the modulation of the VSD-PD coupling mechanism of the KV7.1 channel by the KCNE1 ancillary subunits

The IKS current is diffused through the plasma membranes of cardiomyocytes during the last phase of the cardiac action potential. This repolarization current is conducted by a tetrameric protein complex derived from the co-expression of four voltage-gated potassium channel KV7.1 α-subunits and KCNE1 ancillary subunits from KCNQ1 and KCNE1 genes, respectively. We studied here the conformational space of KV7.1 in presence and absence of KCNE1, by building transmembrane models of their known Resting, Intermediate, and Activated states. We conducted Molecular Dynamics simulations of these models in lipid bilayers including the phosphatidyl-inositol-4,5-bisphosphate (PIP2) lipids. The comparative analysis of MD trajectories obtained for the KV7.1 and IKS models reveals how KCNE1 shifts the coupling mechanism between the activation state of the Voltage Sensor Domain of the channel and the conformation (open or closed) of its Pore Domain.

State-dependent inhibition of BK channels by the opioid agonist loperamide

Large-conductance Ca2+-activated K+ (BK) channels control a range of physiological functions, and their dysfunction is linked to human disease. We have found that the widely used drug loperamide (LOP) can inhibit activity of BK channels composed of either α-subunits (BKα channels) or α-subunits plus the auxiliary γ1-subunit (BKα/γ1 channels), and here we analyze the molecular mechanism of LOP action. LOP applied at the cytosolic side of the membrane rapidly and reversibly inhibited BK current, an effect that appeared as a decay in voltage-activated BK currents. The apparent affinity for LOP decreased with hyperpolarization in a manner consistent with LOP behaving as an inhibitor of open, activated channels. Increasing LOP concentration reduced the half-maximal activation voltage, consistent with relative stabilization of the LOP-inhibited open state. Single-channel recordings revealed that LOP did not reduce unitary BK channel current, but instead decreased BK channel open probability and mean open times. LOP elicited use-dependent inhibition, in which trains of brief depolarizing steps lead to accumulated reduction of BK current, whereas single brief depolarizing steps do not. The principal effects of LOP on BK channel gating are described by a mechanism in which LOP acts as a state-dependent pore blocker. Our results suggest that therapeutic doses of LOP may act in part by inhibiting K+ efflux through intestinal BK channels.