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The Ca 2+ ‐activated Cl − channel TMEM16A(ANO1) modulates, but is not required for, pacemaking in mouse lymphatic vessels
Author(s) -
Gui Peichun,
Zawieja Scott,
Li Min,
Bulley Simon,
Jaggar Jonathan,
Rock Jason,
Davis Michael
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.726.3
Subject(s) - niflumic acid , chemistry , dids , depolarization , membrane potential , lymphatic system , biophysics , pacemaker potential , afterdepolarization , electrophysiology , anatomy , neuroscience , biology , biochemistry , repolarization , immunology , membrane
An unidentified electrical pacemaker within the lymphatic vessel wall initiates the spontaneous contractions of lymphatic muscle (LM) required for normal lymph transport. The current hypothesis for pacemaking is that intracellular Ca 2+ puffs within LM activate a Ca 2+ ‐activated‐Cl − channel (CACC), initiating small depolarizations (STDs) that summate to trigger action potential (APs). The best candidate for the CACC is TMEM16A (ANO1), a channel implicated in pressure‐mediated constriction of vascular smooth muscle. We explored its role in the pacemaking of isolated, pressurized lymphatic vessels from mice, rats and humans. Contractions were always preceded by APs, which were similar in shape among all three species, pointing to a common ionic mechanism. AP firing was preceded not by STDs but by slow, constant diastolic depolarization. Ca 2+ puffs/waves occurred spontaneously in many LM cells, but were not synchronized between cells or correlated with contractions. Ca 2+ entry following the AP spike was mediated by L‐ and T‐type Ca 2+ channels (VGCCs), generating a ~synchronized, global Ca 2+ flash over the entire vessel. SERCA inhibitors abolished puffs, but not flashes or pacemaking, whereas nifedipine abolished flashes and pacemaking. TMEM16A GFP mice expressed GFP in every LM cell, as confirmed by PCR and Western blotting. Whole cell recordings from mouse LM cells revealed an outwardly‐rectifying Ca 2+ ‐activated current, with a reversal potential around zero in symmetric [Cl − ] solutions that shifted with changes in the Cl − equilibrium potential. Both broad‐spectrum (niflumic acid, DIDS) and “selective” (T16A‐01) CACC inhibitors were of limited value as they inhibited VGCCs at concentrations lower than those required to block TMEM16A. We therefore used an inducible tissue‐specific mouse model to delete TMEM16A from LM. SMMHC‐CreERt2;Rosa26 mTmG mice were bred to determine the efficiency of Cre expression in LM, which was >95% in inguinal‐axillary lymphatics and ~99% in popliteal lymphatics. LM cells from SMMHC‐CreERt2;TMEM16A fl/fl mice showed significantly reduced levels of whole‐cell Cl − current compared to TMEM16A fl/fl controls. Spontaneous contractions persisted in pressurized lymphatics from SMMHC‐CreERt2; TMEM16A fl/fl mice, albeit at a significantly reduced frequency, and frequency did not increase normally with pressure elevation. These findings suggest that pacemaking is controlled by the interaction of several LM plasma membrane ionic currents rather than an intracellular Ca 2+ event, but that TMEM16A contributes to a baseline excitatory current. Support or Funding Information NIH R01 HL‐122578