Cyclic nucleotide and kinase inhibitor treatments influence AtPIP2;1 mediated ionic conductance in X. laevis oocytes
AtPIP2;1 expression in X. laevis oocytes elicits currents in the presence of Na+ (Byrt et al., 2017). We investigated whether AtPIP2;1-facilitated ion transport may be altered by its phosphorylation state. The activity of endogenous kinases inX. laevis oocytes, and hence phosphorylation state of expressed proteins, were manipulated by the exogenous application of membrane permeable cyclic nucleotide monophosphate (cNMP) analogues 8-Br-cAMP and 8-Br-cGMP as kinase stimulators, and the kinase inhibitor H7 (Figure 1). These pharmacological agents have been used previously in this heterologous system to manipulate kinase activity for testing the functional regulation of mammalian aquaporins by phosphorylation (Campbell et al., 2012; Han and Patil, 2000; Hoffert et al., 2008; Yool et al., 1996).
Oocytes were injected with water or AtPIP2;1 cRNA and either kept untreated as a control, or incubated in 1 mM 8-Br-cAMP (cAMP) or 8-Br-cGMP (cGMP) for 10 min, or incubated in 10 µM H7 for 2 h, or incubated in H7 prior to a cNMP incubation. The ionic conductance of these oocytes was measured by TEVC (Figure 1). Data was collected from multiple independent oocyte batches; therefore, to remove batch-to-batch variation in native ionic conductance and examine only the response to the treatments the data for treated water injected oocytes were normalised to untreated water injected oocytes (Figure 1a), and treated AtPIP2;1 injected oocytes were normalised to untreated AtPIP2;1 injected oocytes (Figure 1b) within each batch. The representative IV curves of AtPIP2;1 and water injected oocytes are indicated in Figure S1.
Water injected oocytes did not respond to any treatment type, with the exception of a slight increase in conductance that was observed upon cGMP treatment when compared to H2O injected oocytes treated with cAMP (Figure 1a). Incubation of AtPIP2;1 injected oocytes in solutions containing H7 resulted in a significant decrease in ionic conductance relative to untreated (Figure 1b). In contrast, incubation in cAMP increased the ionic conductance of AtPIP2;1 injected oocytes (Figure 1b). AtPIP2;1 injected oocytes that were first incubated in H7 followed by a cAMP incubation had increased ionic conductance compared to those incubated only in H7. This indicates that Na+transport facilitated by AtPIP2;1 in oocytes is potentially influenced by phosphorylation status, assuming that the treatments alter endogenous kinase activity that phosphorylate AtPIP2;1. It has previously been demonstrated that X. laevis can phosphorylate an expressed PIP2 aquaporin (Johansson et al., 1998).