The T‐type Voltage Gated Calcium Channel Cav3.2 is Important for Enteroendocrine Cell Mechanotransduction

Abstract

BACKGROUNDEnteroendocrine cells (EECs) in the gastrointestinal (GI) epithelium are specialized sensors of luminal chemicals and forces. Upon stimulation, EECs release a variety of neurotransmitters and hormones, but cellular mechanisms of excitation‐secretion coupling are poorly understood. We recently discovered that a population of EECs expresses the mechanosensitive ion channel Piezo2, which is necessary for force‐dependent intracellular Ca2+ increase and serotonin release. However, the molecular mechanism that links Piezo2 and serotonin release is unknown. In other sensory systems, the Cacna1h‐encoded t‐type voltage‐gated calcium channel Cav3.2 is important in mechanotransduction for linking mechanical activation with neurotransmitter release.AIMto determine if Cav3.2 is expressed and functionally relevant in EECs.METHODSQuantitative RT‐PCR for Cacna1h mRNA expression was evaluated in colon epithelium of control and NeuroD1 Cre x RiboTag mice, in which actively transcribed mRNA specifically from EECs is tagged with hemagglutinin (HA). NeuroD1 Cre x tdTomato colon cross‐sections were fixed and immunolabeled with anti‐Cav3.2 antibody. Single primary NeuroD1 cre x GCaMP5 EECs were mechanically stimulated and Cav3.2 role in mechanotransduction was tested by pharmacology (Ca2+ free, nickel, mibefradil, ω‐agatoxin IVA) and Cacna1h siRNA knockdown.RESULTSMurine colon epithelium expressed Cav3.2 mRNA. In NeuroD1 Cre x RiboTag mouse, HA co‐localized with EEC marker Chromogranin A (Chga). NeuroD1+ EEC ribosomal trapped mRNA was enriched for markers of epithelium (Vil1), EEC (Chga), and enriched 7.0‐fold for Cav3.2 (n=3, p<0.05 compared to whole epithelium). Immunohistochemistry in NeuroD1 Cre x tdTomato showed that Cav3.2 co‐localized specifically with tdTomato (NeuroD1+) EECs. In primary NeuroD1+ EECs, Cav3.2 inhibition by all pharmacologic agents and siRNA knockdown inhibited mechanically induced Ca2+ response (96±1% for Ca2+ free, 71±9% nickel, 61±6% mibefradil, 80±4% ω‐agatoxin IVA and 96±1% Cav3.2 siRNA, n=3–18, p<0.05 compared to control untreated cells or cells treated with non‐targeted siRNA).CONCLUSIONSCav3.2 is densely and specifically expressed in mouse colon EECs and it is important for regulating Ca2+ in EEC mechanotransduction. Future studies will determine how Cav3.2 channels contribute to EEC electrical excitability, excitation‐secretion coupling, and ultimately EEC Cav3.2 roles in GI physiology.Support or Funding InformationSupported by NIH DK106456, NIH DK084567, and AGA RSAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.