Modulation of TMEM16B channel activity by the calcium-activated chloride channel regulator 4 (CLCA4) in human cells.

The Ca2+-activated Cl- channel regulator CLCA1 potentiates the activity of the Ca2+-activated Cl- channel (CaCC) TMEM16A by directly engaging the channel at the cell surface, inhibiting its reinternalization and increasing Ca2+-dependent Cl- current (ICaCC) density. We now present evidence of functional pairing between two other CLCA and TMEM16 protein family members, namely CLCA4 and the CaCC TMEM16B. Similar to CLCA1, (i) CLCA4 is a self-cleaving metalloprotease, and the N-terminal portion (N-CLCA4) is secreted; (ii) the von Willebrand factor type A (VWA) domain in N-CLCA4 is sufficient to potentiate ICaCC in HEK293T cells; and (iii) this is mediated by the metal ion-dependent adhesion site motif within VWA. The results indicate that, despite the conserved regulatory mechanism and homology between CLCA1 and CLCA4, CLCA4-dependent ICaCC are carried by TMEM16B, rather than TMEM16A. Our findings show specificity in CLCA/TMEM16 interactions and suggest broad physiological and pathophysiological links between these two protein families.

Erdheim-Chester Disease: A Mysterious Illness Causing Obscure Neurologic Syndromes.

Erdheim-Chester disease (ECD) is a very rare form of non-Langerhans histiocytic disorder which affects multiple systems and can present in a variety of different ways. We present two patients diagnosed with ECD whose symptoms, progression and treatment differ dramatically.

Cantu syndrome-associated SUR2 (ABCC9) mutations in distinct structural domains result in KATP channel gain-of-function by differential mechanisms.

The complex disorder Cantu syndrome (CS) arises from gain-of-function mutations in either KCNJ8 or ABCC9, the genes encoding the Kir6.1 and SUR2 subunits of ATP-sensitive potassium (KATP) channels, respectively. Recent reports indicate that such mutations can increase channel activity by multiple molecular mechanisms. In this study, we determined the mechanism by which KATP function is altered by several substitutions in distinct structural domains of SUR2: D207E in the intracellular L0-linker and Y985S, G989E, M1060I, and R1154Q/R1154W in TMD2. We engineered substitutions at their equivalent positions in rat SUR2A (D207E, Y981S, G985E, M1056I, and R1150Q/R1150W) and investigated functional consequences using macroscopic rubidium (86Rb+) efflux assays and patch-clamp electrophysiology. Our results indicate that D207E increases KATP channel activity by increasing intrinsic stability of the open state, whereas the cluster of Y981S/G985E/M1056I substitutions, as well as R1150Q/R1150W, augmented Mg-nucleotide activation. We also tested the responses of these channel variants to inhibition by the sulfonylurea drug glibenclamide, a potential pharmacotherapy for CS. None of the D207E, Y981S, G985E, or M1056I substitutions had a significant effect on glibenclamide sensitivity. However, Gln and Trp substitution at Arg-1150 significantly decreased glibenclamide potency. In summary, these results provide additional confirmation that mutations in CS-associated SUR2 mutations result in KATP gain-of-function. They help link CS genotypes to phenotypes and shed light on the underlying molecular mechanisms, including consequences for inhibitory drug sensitivity, insights that may inform the development of therapeutic approaches to manage CS.

Are the persistent effects of "gate control" stimulation on nociception a form of generalization of habituation that is endocannabinoid-dependent?

Repetitive activation of non-nociceptive afferents is known to attenuate nociceptive signaling. However, the functional details of how this modulatory process operates are not understood and this has been a barrier in using such stimuli to effectively treat chronic pain. The present study tests the hypothesis that the ability of repeated non-nociceptive stimuli to reduce nociception is a form of generalized habituation from the non-nociceptive stimulus-response pathway to the nociceptive pathway. Habituation training, using non-nociceptive mechanosensory stimuli, did reduce responses to nociceptive thermal stimulation. This generalization of habituation to nociceptive stimuli required endocannabinoid-mediated neuromodulation, although disrupting of endocannabinoid signaling did not affect "direct" habituation of to the non-nociceptive stimulus. Surprisingly, the reduced response to nociceptive stimuli following habituation training was very long-lasting (3-8 days). This long-term habituation required endocannabinoid signaling during the training/acquisition phase, but endocannabinoids were not required for post-training retention phase. The implications of these results are that applying principles of habituation learning could potentially improve anti-nociceptive therapies utilizing repeated non-nociceptive stimulation such as transcutaneous nerve stimulation (TENS), spinal cord stimulation (SCS), or electro-acupuncture.

Electrophysiology of human iPSC-derived vascular smooth muscle cells and cell autonomous consequences of Cantu Syndrome mutations.

Objective: Cantu Syndrome (CS), a multisystem disease with a complex cardiovascular phenotype, is caused by GoF variants in the Kir6.1/SUR2 subunits of ATP-sensitive potassium (K ATP ) channels, and is characterized by low systemic vascular resistance, as well as tortuous, dilated vessels, and decreased pulse-wave velocity. Thus, CS vascular dysfunction is multifactorial, with distinct hypomyotonic and hyperelastic components. To dissect whether such complexities arise cell-autonomously within vascular smooth muscle cells (VSMCs), or as secondary responses to the pathophysiological milieu, we assessed electrical properties and gene expression in human induced pluripotent stem cell-derived VSMCs (hiPSC-VSMCs), differentiated from control and CS patient-derived hiPSCs, and in native mouse control and CS VSMCs. Approach and Results: Whole-cell voltage-clamp of isolated aortic and mesenteric VSMCs isolated from wild type (WT) and Kir6.1[V65M] (CS) mice revealed no difference in voltage-gated K + (K v ) or Ca 2+ currents. K v and Ca 2+ currents were also not different between validated hiPSC-VSMCs differentiated from control and CS patient-derived hiPSCs. Pinacidil-sensitive K ATP currents in control hiPSC-VSMCs were consistent with those in WT mouse VSMCs, and were considerably larger in CS hiPSC-VSMCs. Consistent with lack of any compensatory modulation of other currents, this resulted in membrane hyperpolarization, explaining the hypomyotonic basis of CS vasculopathy. Increased compliance and dilation in isolated CS mouse aortae, was associated with increased elastin mRNA expression. This was consistent with higher levels of elastin mRNA in CS hiPSC-VSMCs, suggesting that the hyperelastic component of CS vasculopathy is a cell-autonomous consequence of vascular K ATP GoF. Conclusions: The results show that hiPSC-VSMCs reiterate expression of the same major ion currents as primary VSMCs, validating the use of these cells to study vascular disease. The results further indicate that both the hypomyotonic and hyperelastic components of CS vasculopathy are cell-autonomous phenomena driven by K ATP overactivity within VSMCs.

Complex consequences of Cantu syndrome SUR2 variant R1154Q in genetically modified mice.

Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations in pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunits, the most common mutations being SUR2[R1154Q] and SUR2[R1154W], carried by approximately 30% of patients. We used CRISPR/Cas9 genome engineering to introduce the equivalent of the human SUR2[R1154Q] mutation into the mouse ABCC9 gene. Along with minimal CS disease features, R1154Q cardiomyocytes and vascular smooth muscle showed much lower KATP current density and pinacidil activation than WT cells. Almost complete loss of SUR2-dependent protein and KATP in homozygous R1154Q ventricles revealed underlying diazoxide-sensitive SUR1-dependent KATP channel activity. Surprisingly, sequencing of SUR2 cDNA revealed 2 distinct transcripts, one encoding full-length SUR2 protein; and the other with an in-frame deletion of 93 bases (corresponding to 31 amino acids encoded by exon 28) that was present in approximately 40% and approximately 90% of transcripts from hetero- and homozygous R1154Q tissues, respectively. Recombinant expression of SUR2A protein lacking exon 28 resulted in nonfunctional channels. CS tissue from SUR2[R1154Q] mice and human induced pluripotent stem cell-derived (hiPSC-derived) cardiomyocytes showed only full-length SUR2 transcripts, although further studies will be required in order to fully test whether SUR2[R1154Q] or other CS mutations might result in aberrant splicing and variable expressivity of disease features in human CS.

Coronavirus Proteins as Ion Channels: Current and Potential Research.

Coronavirus (CoV) outbreaks have recently emerged as a global public health threat due to their exceptional zoonotic potential - a feature arising from their ability to infect a diverse range of potential hosts combined with their high capacity for mutation and recombination. After Severe Acute Respiratory Syndrome (SARS) CoV-1 in 2003 and Middle East Respiratory Syndrome (MERS) CoV in 2012, with the current SARS-CoV-2 pandemic we are now in the midst of the third deadly international CoV outbreak in less than 20 years. Coronavirus outbreaks present a critical threat to global public health and an urgent necessity for therapeutic options. Here, we critically examine the current evidence for ion channel activity in CoV proteins and the potential for modulation as a therapeutic approach.

UBXN2A enhances CHIP-mediated proteasomal degradation of oncoprotein mortalin-2 in cancer cells.

Overexpression of oncoproteins is a major cause of treatment failure using current chemotherapeutic drugs. Drug-induced degradation of oncoproteins is feasible and can improve clinical outcomes in diverse types of cancers. Mortalin-2 (mot-2) is a dominant oncoprotein in several tumors, including colorectal cancer (CRC). In addition to inactivating the p53 tumor suppressor protein, mot-2 enhances tumor cell invasion and migration. Thus, mot-2 is considered a potential therapeutic target in several cancer types. The current study investigated the biological role of a ubiquitin-like protein called UBXN2A in the regulation of mot-2 turnover. An orthogonal ubiquitin transfer technology followed by immunoprecipitation, in vitro ubiquitination, and Magnetic Beads TUBE2 pull-down experiments revealed that UBXN2A promotes carboxyl terminus of the HSP70-interacting protein (CHIP)-dependent ubiquitination of mot-2. We subsequently showed that UBXN2A increases proteasomal degradation of mot-2. A subcellular compartmentalization experiment revealed that induced UBXN2A decreases the level of mot-2 and its chaperone partner, HSP60. Pharmacological upregulation of UBXN2A using a small molecule, veratridine (VTD), decreases the level of mot-2 in cancer cells. Consistent with the in vitro results, UBXN2A+/- mice exhibited selective elevation of mot-2 in colon tissues. An in vitro Anti-K48 TUBE isolation approach showed that recombinant UBXN2A enhances proteasomal degradation of mot-2 in mouse colon tissues. Finally, we observed enhanced association of CHIP with the UBXN2A-mot-2 complex in tumors in an azoxymethane/dextran sulfate sodium-induced mouse CRC model. The existence of a multiprotein complex containing UBXN2A, CHIP, and mot-2 suggests a synergistic tumor suppressor activity of UBXN2A and CHIP in mot-2-enriched tumors. This finding validates the UBXN2A-CHIP axis as a novel and potential therapeutic target in CRC.