Acid-sensing ion channels (ASICs) are trimeric cation channels that undergo activation and desensitization in response to extracellular acidification. The underlying mechanism coupling proton binding in the extracellular region to pore gating is unknown. Here we probed the reactivity toward methanethiosulfonate (MTS) reagents of channels with cysteine-substituted residues in the outer vestibule of the pore of ASIC1a. We found that positively-charged MTS reagents trigger pore opening of G428C. Scanning mutagenesis of residues in the region preceding the second transmembrane spanning domain indicated that the MTSET-modified side chain of Cys at position 428 interacts with Tyr-424. This interaction was confirmed by double-mutant cycle analysis. Strikingly, Y424C-G428C monomers were associated by intersubunit disulfide bonds and were insensitive to MTSET. Despite the spatial constraints introduced by these intersubunit disulfide bonds in the outer vestibule of the pore, Y424C-G428C transitions between the resting, open, and desensitized states in response to extracellular acidification. This finding suggests that the opening of the ion conductive pathway involves coordinated rotation of the second transmembrane-spanning domains. Acid-sensing ion channels (ASICs)2 are voltage-independent ligand-gated ion channels expressed throughout neurons of the mammalian central and peripheral nervous systems (1, 2). ASICs are members of the Epithelial Sodium Channel/Degenerin (ENaC/Deg) family, a large group of proteins that are implicated in mechanosensation, pain sensation, regulation of extracellular fluid volume, and airway surface liquid volume (2-5). These channels are organized as homo-or hetero-trimers. Each subunit has two transmembrane segments (TMs) connected by a large extracellular region with the N and C termini on the intracellular side (6, 7). ASICs are constitutively closed and undergo activation and desensitization in response to extracellular acidification. Seven ASIC subunits are expressed in mammals: ASIC1a, ASIC1b, ASIC1b2, ASIC2a, ASIC2b, ASIC3, and ASIC4 (2). The sensitivity of activation and desensitization by protons depend on the specific subunits forming the channel complex.The pore of ASIC1 in the presumed desensitized-like state has an hourglass-like shape defined mainly by residues from TM2, although residues from TM1 form a portion of the outer vestibule (6, 7). The TM2 helices are tilted by a ϳ50°angle from the membrane normal, and their intersection likely constitutes the desensitization gate. The TM1 segments are in contact with the lipid bilayer and with the TM2 segments in their own and neighboring subunits. Compelling evidence suggests that ASIC activation involves protonation of multiple residues within the extracellular region of the protein (6, 8 -10). A fundamental question regarding the mechanism of gating of ENaC/Deg channels is how extracellular cues trigger pore opening and closing events. The extracellular region of ASIC1 is organized in discrete subdomains referred to as the palm, ...

Na(+) absorption and K(+) secretion in the distal segments of the nephron are modulated by the tubular flow rate. Epithelial Na(+) channels (ENaC), composed of α-, β-, and γ-subunits respond to laminar shear stress (LSS) with an increase in open probability. Higher vertebrates express a δ-ENaC subunit that is functionally related to the α-subunit, while sharing only 35% of sequence identity. We investigated the response of δβγ channels to LSS. Both the time course and magnitude of activation of δβγ channels by LSS were remarkably different from those of αβγ channels. ENaC subunits have similar topology, with an extracellular region connected by two transmembrane domains with intracellular N and C termini. To identify the specific domains that are responsible for the differences in the response to flow of αβγ and δβγ channels, we generated a series of α-δ chimeras and site-specific α-subunit mutants and examined parameters of activation by LSS. We found that specific sites in the region encompassing and just preceding the second transmembrane domain were responsible for the differences in the magnitude and time course of channel activation by LSS.

Background Molecular testing of thyroid nodules with indeterminate fine‐needle aspiration (FNA) cytology is commonly used to guide patient management and is typically performed on freshly collected FNA samples. In this study, the authors evaluated the performance of the ThyroSeq test in cytology smear slides. Methods Air‐dried Diff‐Quik (DQ)‐stained and alcohol‐fixed Papanicolaou (Pap)‐stained smears were used to determine required cellularity and sensitivity of mutation detection and to compare ThyroSeq v3 Genomic Classifier (GC) results obtained in cytology smears and fresh FNA samples from the same nodules. Results ThyroSeq testing of 31 cytology smears revealed that 25 smears (81%) were adequate for ThyroSeq analysis, including 14 Pap‐stained smears (100%) and 11 DQ‐stained smears (65%), whereas 6 DQ‐stained smears (35%) failed RNA sequencing. The overall accuracy for detecting molecular alterations was 98%, with 100% concordance for mutations and gene expression alterations, 96% concordance for fusions, and 94% concordance for copy number alterations. Cytology smears were adequate for ThyroSeq analysis when at least 200 to 300 cells were present in 1 to 3 slides. ThyroSeq detected all studied mutations down to 5% allele frequency and BRAF mutations down to 1% allele frequency. Testing of smears yielded a positive ThyroSeq GC result in all nodules originally classified as positive. Conclusions Thyroid FNA cytology smear slides with adequate cellularity can be successfully used for ThyroSeq GC testing in approximately 80% of cases, with an even higher success rate in Pap‐stained smears. Compared with FNA samples collected into preservative solution, 94% to 100% of different genetic alterations could be accurately detected in smears, validating cytology smears as an alternative for ThyroSeq testing in patients with indeterminate thyroid cytology.

Fine-needle aspiration (FNA) of thyroid nodules yields indeterminate cytological diagnosis in ~20% of cases, confounding patient management. This includes Hurthle cell nodules, which typically yield Bethesda IV and III cytology. Chromosomal copy number alterations (CNA) are known to occur in thyroid tumors, particularly in Hurthle cell carcinomas (HCC) as well as in other typically follicular-patterned tumors including papillary thyroid carcinomas (PTC) and poorly differentiated thyroid carcinomas (PDTC). The aim of this study was to evaluate thyroid nodules tested positive for CNA but negative for all other genomic alterations using ThyroSeq v3 NGS assay in order to establish the probability of cancer in these nodules and find whether it is influenced by the pattern of CNA and nodule size. We evaluated 111 nodules with multiple CNA detected by ThyroSeq in FNA samples and available surgical pathology outcome. Of those, 69 (62%) nodules showed CNA changes consistent with genome near-haploidization (GNH) whereas 42 (38%) nodules had multiple chromosomal losses and gains (CLG). Nodule size ranged from 0.5-10.2 cm; cytology was Bethesda III in 54%, Bethesda IV in 43%, and Bethesda V-VI in 3% of cases, with Hurthle cells mentioned in the cytology report in 64% of cases. On surgical pathology, 38 (34%) of these nodules were malignant (including 24 HCC, 8 PTC, and 5 oncocytic PDTC) and 73 (66%) were benign (including 43 Hurthle cell and 18 follicular adenomas). No significant difference was observed in probability of malignancy between the two patterns of CNA (p=0.41). However, a significant correlation between the nodule size and probability of cancer was found (p=0.006). In specific CNA groups, correlation between cancer and nodule size remained significant for nodules with GNH pattern (P=0.0002), but not with CLG pattern (p=0.449). Specifically, cancer probability in nodules with GNH pattern and <2 cm in size was 14% (all cancers minimally-invasive), 2.0-2.9 cm was 33%, 3.0-3.9 cm was 50%, 4-4.9 cm was 67%, and ≥5 cm was 80%. Among high-risk cancers (widely-invasive or angioinvasive HCC, PDTC), all 10 tumors had the GNH pattern (p=0.01) and average nodule size of 4.9 cm (range, 2.1-8.5 cm). These findings suggest that CNA of both types are frequently found in Hurthle cell tumors, and probability of cancer in nodules with CNA and no other mutations increases with larger nodule size. This may help to refine the pre-operative assessment of cancer probability and risk of more aggressive disease and offer more tailored management to these patients.

Background: Chromosomal rearrangements leading to gene fusions are a known molecular mechanism of thyroid cancer. ThyroSeq Genomic Classifier (GC) allows detecting most of the known fusion types preoperatively by analyzing thyroid fine-needle aspiration (FNA) samples collected from thyroid nodules, but it also facilitates discovery of novel fusion types. The goal of this study was to evaluate the prevalence and types of known and novel fusions detected in thyroid nodules during routing clinical testing and establish histopathologic and clinical correlations. Design: Overall, 6,800 consecutive thyroid nodules were tested by a targeted next-generation sequencing assay (ThyroSeq v3 GC) using material collected by fine-needle aspiration (FNA). In addition, 17 surgically removed tumors were tested for confirmation of fusion subtype. Surgical pathology and clinical follow-up was obtained when available. Immunohistochemistry was performed on selected cases. Results: Gene fusions were identified in 318 (4.68%) of consecutively tested thyroid nodules. The most common fusion type was THADA/IGF2BP3 detected in 121 (38%) cases, followed by RET/PTC in 52 (16%), NTRK3 in 47 (15%), and PPARG in 47 (15%). Other fusions were found at lower prevalence, including BRAF in 14 (4%), ALK in 9 (3%), THADA/TRA2A in 8 (3%), and NTRK1 in 8 (3%) cases. In addition, 12 (4%) of nodules had rare and novel fusions, including PAX8/GLIS fusions. Presence of PAX8/GLIS fusions was confirmed in resected tumor samples. Surgical pathology information was collected on 49 (16%) positive cases and is ongoing. RET/PTC, NTRK3, NTRK1 , and ALK fusions were all diagnosed as classic papillary thyroid carcinoma (PTC) or follicular variant of PTC. All 3 ALK -positive PTC and 5 THADA/IGF2BP3 NIFTP showed strong and diffuse immunoreactivity with ALK and IMP3 antibodies, respectively. THADA/IGF2BP3 was found in noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) (50%), PTC (40%) and follicular thyroid carcinoma (FTC) (10%). PPARG fusions were found in FTC (25%), Hurthle cell carcinoma (25%), PTC (25%) and follicular adenomas (25%). One BRAF fusion ( SND1 / BRAF ) was found in follicular adenoma and all other nodules were PTC. All PAX8 / GLIS fusions were characteristic of hyalinizing trabecular tumors. Conclusions : ThyroSeq GC detected gene fusions in approximately 5% of thyroid nodules that underwent FNA. ThyroSeq overall correct call rate for fusion...