The α 1H Ca2+ channel subunit is expressed in mouse jejunal interstitial cells of Cajal and myocytes

T-type Ca2+ currents have been detected in cells from the external muscular layers of gastrointestinal smooth muscles and appear to contribute to the generation of pacemaker potentials in interstitial cells of Cajal from those tissues. However, the Ca2+ channel subunit responsible for these currents has not been determined. We established that the α subunit of the α1H Ca2+ channel is expressed in single myocytes and interstitial cells of Cajal using reverse transcription and polymerase chain reaction from whole tissue, laser capture microdissected tissue and single cells isolated from the mouse jejunum. Whole-cell voltage clamp recordings demonstrated that a nifedipine and Cd2+ resistant, mibefradil-sensitive current is present in myocytes dissociated from the jejunum. Electrical recordings from the circular muscle layer demonstrated that mibefradil reduced the frequency and initial rate of rise of the electrical slow wave. Gene targeted knockout of both alleles of the cacna1h gene, which encodes the α 1H Ca2+ channel subunit, resulted in embryonic lethality because of death of the homozygous knockouts prior to E13.5 days in utero. We conclude that a channel with the pharmacological and molecular characteristics of the α 1H Ca2+ channel subunit is expressed in interstitial cells of Cajal and myocytes from the mouse jejunum, and that ionic conductances through the α 1H Ca2+ channel contribute to the upstroke of the pacemaker potential. Furthermore, the survival of mice that do not express the α 1H Ca2+ channel protein is dependent on the genetic background and targeting approach used to generate the knockout mice

Oncogenic gene expression and epigenetic remodeling of cis-regulatory elements in ASXL1-mutant chronic myelomonocytic leukemia

Myeloid neoplasms are clonal hematopoietic stem cell disorders driven by the sequential acquisition of recurrent genetic lesions. Truncating mutations in the chromatin remodeler ASXL1 (ASXL1MT) are associated with a high-risk disease phenotype with increased proliferation, epigenetic therapeutic resistance, and poor survival outcomes. We performed a multi-omics interrogation to define gene expression and chromatin remodeling associated with ASXL1MT in chronic myelomonocytic leukemia (CMML). ASXL1MT are associated with a loss of repressive histone methylation and increase in permissive histone methylation and acetylation in promoter regions. ASXL1MT are further associated with de novo accessibility of distal enhancers binding ETS transcription factors, targeting important leukemogenic driver genes. Chromatin remodeling of promoters and enhancers is strongly associated with gene expression and heterogenous among overexpressed genes. These results provide a comprehensive map of the transcriptome and chromatin landscape of ASXL1MT CMML, forming an important framework for the development of novel therapeutic strategies targeting oncogenic cis interactions

Distribution and Characterization of ANO10 in Mouse Small Intestine

Background: Anoctamin 10 (Ano10) belongs to the class of proteins that includes Ca2+ activated Cl-channels such as Ano1, which is expressed in interstitial cells of Cajal (ICC). Mutations in Ano10 lead to spinocerebellar ataxia and immunological defects. Evidence suggests that Ano10 is not a Ca2+ -activated Cl-channel but rather an intracellular protein that may regulate intracellular Ca2+. The aim of this study was to characterize the distribution of Ano10 in the mouse small intestine in order to determine the cell types in which Ano10 may contribute to cellular physiology. Methods: Small intestinal tissues from adult Balb/c (n=3) mice were obtained for both cryosections (10 μm thickness) and whole mount preparations. Tissues were immunolabeled using antibodies raised against Ano10 (rabbit polyclonal), a marker of ICC - Kit (goat polyclonal), a pan-neuronal marker - HuC/D (human, serum derived), and a macrophage marker - F4/80 (rat monoclonal). Secondary antibody controls were performed by exposing samples to secondary antibody in the absence of primary antibody to test for non-specific staining. Double labeling of Ano10 with other cell specific markers was performed on 5 cryosections and 2 whole mounts for each mouse. The distribution of immunoreactivity (IR) was determined by confocal microscopic imaging. Results: Ano10-IR was found to be present in neuronal cell bodies outside of the nucleus. Ano10-IR was present in 100% of HuC/D-positive neurons of the myenteric and submucosal plexuses. Ano10-IR was not found in ICC of the myenteric region; however, there was colocalization of Ano10-IR with Kit-IR on ICC of the deep muscularis plexus (ICC-DMP). Ano10-IR also colocalized with F4/80-IR present on resident macrophages in the region of the myenteric plexus. 100% of F4/80-IR colocalized with Ano10-IR. Conclusions: Immuno-reactivity for Ano10 is present in all types of myenteric and submucosal neurons and in all resident macrophages. Ano10-IR was present in a subset of ICC, specifically ICC-DMP but not ICC in the myenteric plexus. Given the putative role of Ano10 as a regulator of intracellular Ca2+, Ano10 may play a role both in regulation of gastrointestinal motility via neurons and ICC-DMP, as well as immunological responses via resident macrophages. Grant support: NIH DK057061, P01DK68055, and P30DK08456

Non-canonical translation start sites in the TMEM16A chloride channel

TMEM16A is a plasma membrane protein with voltage- and calcium-dependent chloride channel activity. The role of the various TMEM16A domains in expression and function is poorly known. In a previous study, we found that replacing the first ATG of the TMEM16A coding sequence with a nonsense codon (M1X mutation), to force translation from the second ATG localized at position 117, only had minor functional consequences. Therefore, we concluded that this region is dispensable for TMEM16A processing and channel activity. We have now removed the first 116 codons from the TMEM16A coding sequence. Surprisingly, the expression of the resulting mutant, Δ(1-116), resulted in complete loss of activity. We hypothesized that, in the mutant M1X, translation may start at a position before the second ATG, using a non-canonical start codon. Therefore, we placed an HA-epitope at position 89 in the M1X mutant. We found, by western blot analysis, that the HA-epitope can be detected, thus demonstrating that translation starts from an upstream non-ATG codon. We truncated the N-terminus of TMEM16A at different sites while keeping the HA-epitope. We found that stepwise shortening of TMEM16A caused an in parallel stepwise decrease in TMEM16A expression and function. Our results indicate that indeed the N-terminus of TMEM16A is important for its activity. The use of an alternative start codon appears to occur in a naturally-occurring TMEM16A isoform that is particularly expressed in human testis. Future experiments will need to address the role of normal and alternative amino-terminus in TMEM16A structure and function