University of Pittsburgh Department of Biological Sciences Presents:
Friday Noon Seminar Series 2017-2018
Graduate Student: Maiwase Tembo
Signaling Mechanisms of the Calcium Activated Chloride Channel TMEM16a
Transmembrane member 16A (TMEM16a) is a widely expressed Ca2+- activated Cl- channel with various physiological functions ranging from mucosal secretion to regulating smooth muscle contraction. Despite its importance, we are just beginning to understand the mechanisms that regulate TMEM16a gating. Here we recorded Ca2+-evoked Cl- currents passed by the endogenous TMEM16a channels expressed in oocytes from the African clawed frog, Xenopus laevis. Using the inside-out configuration of the patch clamp technique, we have found that the TMEM16a-conducting currents run down within a few seconds of patch excision, despite the continued presence of Ca2+. Current rundown is common amongst channels regulated by phosphatidylinositol 4,5-bisphosphate (PIP2). Thus, we tested the hypothesis that TMEM16a is potentiated by PIP2 using inside-out patches exposed to PIP2 sequestering and recovering agents. First, we reasoned that if PIP2 disassociation with TMEM16a is caused by dephosphorylation, application of Mg-ATP should maintain the phosphorylation status of PIP2 and slow current rundown, and we observed that it did. Next, we found that application of the PIP2 sequestering agents, neomycin and anti-PIP2, to the intracellular surface of the patch sped TMEM16a current rundown by two-fold. In another series of experiments, we sought to recover TMEM16a current after rundown with PIP2 application. We applied the soluble dioctanoyl-PIP2 analog (diC8-PIP2) with Ca2+ and observed greater than 40% TMEM16a current recovery. Conversely, application of soluble dioctanoyl-phosphatidyl inositol (diC8-PI), the backbone of PIP2 without the two phosphate groups, was not able to recover current, nor was diC8-PIP2 applied with no Ca2+. Taken together, our data demonstrate that TMEM16a requires both Ca2+ and PIP2 to pass current.
Friday, December 8, 2017
A219B Langley Hall
12:00 PM Seminar