MCDB seminar will be from Crystal Lara-Santos from the Carlson lab will be presenting their research titled:
Title: Lipid Modulation of Ion Channels: A Novel Calcium-Activated Chloride Channel TMEM16A Regulation Switch?
Friday, March 28, 2025
A219B Langley Hall
12:00 PM
Ion channels are essential for maintaining cellular homeostasis, facilitating electrical signaling, and regulating ion flux across membranes, making them critical for processes, such as muscle tone, inflammation, and fluid secretion. Among these, Transmembrane protein 16A (TMEM16A) is a calcium-activated chloride channel that plays a role in chloride conductance involving the regulation of various physiological processes. In eggs from the African claw frog, Xenopus laevis, fertilization opens TMEM16A channels in a process that stops multiple sperm from entering a fertilized egg. In humans, TMEM16A is associated with the cardiovascular system, controlling muscle contraction by regulating blood pressure. As a calcium-activated chloride channel it requires calcium to open provoking a Cl- efflux in response to the active state. Beyond calcium, emerging evidence suggests that lipid interaction, particularly lipids found in the membrane, significantly impacts its regulation. One of the TMEM16A channel’s key regulators is the acidic phospholipid, PIP2, which potentiates channel activation by stabilizing its open state. Given that PIP2 consists of a phosphorylated inositol head group and two acyl tails, arachidonyl and stearyl we are interested in exploring whether free fatty acids, particularly polyunsaturated fatty acids (PUFAs), regulate TMEM16A. Using excised patches from X. laevis oocytes, natively expressing TMEM16A, we examined how with the presence of calcium, PUFAs with distinct structural properties affect TMEM16A-conducted currents after application, using the inside-out patch clamp technique. Our findings reveal that PUFAs inhibit TMEM16A currents. Specifically, 10 µM α-linolenic acid (C18:3) reduced TMEM16A currents by 53 ± 6% (N=5), while 10 µM arachidonic acid (AA) (C20:4) reduced them by 82 ± 2% (N=9). In contrast, saturated stearic acid (SA) (C18:0) only inhibited 9 ± 6% (N=5), indicating that unsaturated bonds are essential for inhibition. The length of the acyl tail was not critical, as stearidonic acid (C18:4) inhibited 79 ± 6% of the current (N=6), comparable to AA. Additionally, the position of the double bonds did not affect inhibition; ω-3 and ω-6 AA (C20:4) similarly inhibited currents by 75 ± 3% and 82 ± 2%, respectively (N=5-9). Moreover, AA with a less negatively charged methyl-ester and glycine head group inhibited 83 ± 2% (N=5), whereas AA with a bulky head group taurine showed no inhibition (0.1 ± 3%, N=5), suggesting bulkier head groups may interfere with the inhibitory effect on TMEM16A. I suspect that AA’s ability to inhibit TMEM16A currents is driven by its ability to outcompete PIP2, thereby interfering with PIP2 -mediated activation of the channel. To test this hypothesis, I applied different concentrations of PIP2 to excised patches pretreated with 1 µM AA, aiming to stop the inhibition or recover TMEM16A currents. Our results showed that while PIP2 attenuated AA inhibitory effects, it did not fully recover TMEM16A-conducted currents. This suggests that AA inhibitory mechanisms can possibly be driven by perturbing PIP2 ability to potentiate TMEM16A channels. These insights into TMEM16A-PUFA interactions are essential for understanding the regulatory mechanisms of these channels in native cells. Moreover, delineating how PUFAs alter TMEM16A activity may provide a mechanistic link, revealing how AA might regulate TMEM16A channels in vivo and how diets rich in PUFAs beneficially impact many physiological processes.