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bioRxiv · 10.1101/030296

Reconstitution of CO2 regulation of SLAC1 anion channel and function of CO2-permeable PIP2;1 aquaporin as carbonic anhydrase 4 interactor

Cun Wang·Honghong Hu·Xue Qin·Brian Zeise·Danyun Xu·Wouter-Jan Rappel·Walter F Boron·Julian I Schroeder
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Abstract

Daily dark periods cause an increase in the leaf CO2 concentration (Ci) and the continuing atmospheric [CO2] rise also increases Ci. Elevated Ci causes closing of stomatal pores thus regulating gas exchange of plants. The molecular signaling mechanisms leading to CO2-induced stomatal closure are only partially understood. Here we demonstrate that high intracellular [Formula] enhances currents mediated by the guard cell S-type anion channel SLAC1 when co-expressing either of the protein kinases OST1, CPK6 or CPK23 in Xenopus oocytes. Split-ubiquitin screening identified the PIP2;1 aquaporin as an interactor of the {beta}CA4 carbonic anhydrase, which was confirmed in split luciferase, bimolecular fluorescence complementation and co-immunoprecipitation experiments. PIP2;1 exhibited CO2 permeability. Co-expression of {beta}CA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1 enabled extracellular CO2 enhancement of SLAC1 anion channel activity. An inactive PIP2;1 point mutation was identified which abrogated water and CO2 permeability and extracellular CO2 regulation of SLAC1 activity in Xenopus oocytes. These findings identify the CO2-permeable PIP2;1 aquaporin as key interactor of carbonic anhydrases, show functional reconstitution of extracellular CO2 signaling to ion channel regulation and implicate SLAC1 as a bicarbonate-responsive protein in CO2 regulation of S-type anion channels.

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Cun Wang, Honghong Hu, Xue Qin, Brian Zeise, Danyun Xu, Wouter-Jan Rappel, Walter F Boron, Julian I Schroeder. 2015-11-10. Reconstitution of CO2 regulation of SLAC1 anion channel and function of CO2-permeable PIP2;1 aquaporin as carbonic anhydrase 4 interactor. https://doi.org/10.1101/030296

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