Objective: In Ehrlich Ascites Tumor cells (EATC) TASK-2 is known to be the dominant volume regulated K⁺ channel (Hoffmann et al. 2009). The purpose of the present study is to investigate the regulation of TASK-2 both after short and long term hypotonic stimulation. Methods: The specific activity of tyrosine phosphorylation on the TASK-2 channel was measured after short term hypotonic stimulation (1, 5 and 10 min) using antibody against TASK-2 and the phospho-specific antibody pY100. The potassium current through the TASK-2 channel was measured using patch clamp technique and cells were kept in isotonic solution (300 mOsm) or hypotonic solution (180 mOsm). Measurement was carried out after 24 and 48 hours. TASK-2 mRNA levels were measured using micro array analysis (48h) and real-time PCR. Through western blotting we will furthermore study the amount of TASK-2 in cells after long term hypotonicity (12, 24 and 48 h). Results: Short term hypotonicity resulted in a significantly increased and time dependent tyrosine phosphorylation of the TASK-2 channel itself. Long term hypotonicity (24 and 48 h) resulted in a significant decreased current through the volume sensitive TASK-2 channel after 48 hours and a decreased, though not significant, current after 24 hours, compared to isotonic control. Micro array analysis and real- time PCR analysis of the TASK-2 mRNA levels showed a down-regulation when long term hypotonically stimulated. Conclusions: Activation of the TASK-2 channel by short term hypotonicity requires a tyrosine phosphorylation upon the channel itself whereas long term hypotonicity results in a down-regulation of the TASK-2 channel on gene level. Through further studies we will investigate the result of long term hypotonicity on protein levels and identify the amino acids that are tyrosine phosphorylated after short term hypotonic stimulus.

Hoffmann, E.K., Lambert, I.H. & Pedersen S.F. 2009. Physiol Rev 89(1):193-277.