PIP2 and TMEM16A

By comparing the proportion of peak current decay at +60 and -60 mV, we found that the current decay was not voltage dependent. By comparing the proportion of current decay with the peak current size of individual patches, we found that the rate of current decay was similar between patch recordings regardless of applied voltage or the total current.

Rundown of current conducted by channels recorded from in excised inside-out patches is characteristic of channels regulated by the acidic lipid PIP2. We therefore hypothesized that if TMEM16A current rundown occurred due to the depletion of PIP2 in excised patches, PIP2 application should recover TMEM16A currents following current decay. We tested this hypothesis by applying dicotanoylglycerol-PIP2 (diC8-PIP2), a water-soluble analog of PIP2, to inside-out patches excised from X. laevis oocytes. For these experiments, we recorded TMEM16A-conducted currents before and during application of 2 mM Ca2+. Once the Ca2+-activated currents ran down to a steady state, we applied 100 μM diC8-PIP2 in the presence of 2 mM Ca2+. We observed that 100 μM diC8-PIP2 application recovered 3.6 +/- 0.5-fold current. We next determined if diC8-PIP2 was sufficient to recover TMEM16A current following rundown, or if diC8-PIP2 and Ca2+ are both required. We found that diC8-PIP2 application in the absence of Ca2+ had a nominal effect on TMEM16A, changing the current by 1.1 +/- 0.1-fold. These data demonstrate that although Ca2+ activates TMEM16a, PIP2 is required for these channels to conduct Cl- currents. Moreover, PIP2 potentiates TMEM16A currents only in the presence of intracellular Ca2+.

The diC8-PIP2 compound could theoretically regulate TMEM16A channels by interactions mediated by its lipid tail group, or its phosphoinositol head group. Thus, we tested the hypothesis that the dicotanoylglycerol-phosphoinositol backbone (diC8-PI) alone was sufficient to recover current. Following TMEM16A current rundown, we applied 100 μM diC8-PI with Ca2+ and quantified the proportion of current recovered. We observed that 100 μM diC8-PI application nominally altered the TMEM16A currents, recovering 1.4 +/- 0.2-fold current (1-fold recovery represents no change in current). This result demonstrates that without the phosphorylated head groups, diC8-PI was unable to recover TMEM16A currents suggesting that the phosphates mediate the TMEM16a-PIP2 interaction. Altogether, the data reveal that in addition to Ca2+, PIP2 regulates TMEM16A gating and is required for these channels to conduct current.

Our finding that diC8-PIP2 restored TMEM16A currents, suggested that rundown may be the result of PIP2 depletion in excised inside-out patches. Thus, we reasoned that we should be able to speed TMEM16A current rundown by applying compounds that compete with TMEM16A for binding to PIP2. We tested this hypothesis by determining whether application of two compounds known to scavenge PIP2, altered the rate of TMEM16A current decay: a PIP2-targeting antibody (anti-PIP2) or neomycin. Indeed, we observed that both anti-PIP2 and neomycin sped TMEM16A current rundown.

PIP2 can be depleted through its dephosphorylation of the inositol head group and recreated through re-phosphorylation of the PI. Within an intact cell, these phosphatases and kinases together act to maintain a stable concentration of PIP2. However, in excised patches, these membrane-anchored kinases lack access to the ATP required to fuel re-phosphorylation and regeneration of PIP2. In other words, phosphatases can still dephosphorylate PIP2, but the kinases cannot rephosphorylate the lipid. We found that application of Mg-ATP along with Ca2+ to excised patches, which should enable resphosphorylation of PIP2, slowed TMEM16A current rundown in excised inside-out patches.

Next, we reasoned that if phosphatase-mediated PIP2 depletion causes TMEM16A current rundown in excised patches then inhibiting phosphatase activity would also slow rundown. Indeed we found that in application of Ca2+ with the general phosphatase inhibitor sodium beta-glycerophosphate pentahydrate (BGP), slowed TMEM16A rundown. These data suggest that TMEM16A currents rundown in excised patches as the result of PIP2 depletion via its dephosphorylation.