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).