Crystal Lara and Katherine Sharp to Speak

Crystal Lara of the Carlson lab and Katherine Sharp of the Brodsky lab to speak

Friday, September 23rd, 2022

A219B Langley Hall

12:00 PM


Is Ca2+ just a TMEM16A activator? (Crystal)

Investigating the fate of misfolded membrane proteins in the mammalian ER (Katherine)



Transmembrane protein 16A (TMEM16A) is a Ca2+-activated Cl- channel that regulates several physiological processes in humans and diverse animals alike. Previous studies have shown that TMEM16A channels are expressed in the cardiovascular system where they controlling smooth muscle contraction thereby playing a commanding role in regulating blood pressure. In animals like the African clawed frog, Xenopus laevis, fertilization activates TMEM16A channels and induces the block of the entrance of additional sperm to a fertilized egg. Despite the robust evidence that this channel regulates diverse and critical physiological processes, little is known about how this channel is regulated by signals other than Ca2+. Recently, the Carlson lab has established that in addition to Ca2+, TMEM16A channels require the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to open. Here, we used excised inside-out patches to explore the relationship between intracellular Ca2+ and TMEM16A regulation. Like other channels regulated by PI(4,5)P2, TMEM16A-conducted currents recorded in excised patches decayed shortly after patch excision. Here we found that the concentration of applied Ca2+ altered the rate of rundown, with high Ca2+ concentrations speeding rundown by activating membrane associated phospholipase C (PLC). Together, these results clarify our understanding of how Ca2+ regulates both TMEM16A directly, and targets PLC to regulate the membrane PI(4,5)P2 content.


Approximately one-third of all newly synthesized proteins enter the secretory pathway at the endoplasmic reticulum (ER) where they undergo conformational changes to adopt a functional structure. To maintain homeostasis, terminally misfolded proteins are often retained in the ER and degraded by ER-associated degradation (ERAD). ER chaperones stabilize proteins during folding and are sufficient for the retention of soluble misfolded proteins. In contrast, retention of misfolded proteins within the ER membrane—at least in yeast—is partially dependent on aggregation propensity and ubiquitination. Alternatively, misfolded proteins may escape the ER and be degraded by the vacuole/lysosome. Previous work in the Brodsky lab utilized a model substrate, SZ*, that has characteristics amenable to both fates and found that a ubiquitin-dependent ER retention factor, Ubx2, retains SZ* for ERAD in yeast. Whether the human homologues of Ubx2 play a similar role remains unclear. I hypothesize that two mammalian homologues, UBXD2 and UBXD8, similarly promote retention of ERAD substrates. To address this hypothesis, I conducted cycloheximide chase assays to assess the degradation of SZ* in HEK293 cells. I found that degradation of SZ* is proteasome-dependent, consistent with the ERAD pathway. I will examine the fate of SZ* after siRNA-mediated knockdown of UBXD2 and UBXD8 to determine whether these are contributing to ER retention. This investigation will be extended to additional substrates such as the cystic fibrosis transmembrane conductance regulator (CFTR). Understanding mechanisms of ER retention for misfolded membrane proteins will provide valuable insights into cellular decision making in the context of human diseases.


23 Sep 2022

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A219B Langley Hall