Antagonistic regulation of swelling-activated Cl−current in rabbit ventricle by Src and EGFR protein tyrosine kinases

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Abstract
Regulation of swelling-activated Cl−current ( ICl,swell) is complex, and multiple signaling cascades are implicated. To determine whether protein tyrosine kinase (PTK) modulates ICl,swelland to identify the PTK involved, we studied the effects of a broad-spectrum PTK inhibitor (genistein), selective inhibitors of Src (PP2, a pyrazolopyrimidine) and epidermal growth factor receptor (EGFR) kinase (PD-153035), and a protein tyrosine phosphatase (PTP) inhibitor (orthovanadate). ICl,swellevoked by hyposmotic swelling was increased 181 ± 17% by 100 μM genistein, and the genistein-induced current was blocked by the selective ICl,swellblocker tamoxifen (10 μM). Block of Src with PP2 (10 μM) stimulated tamoxifen-sensitive ICl,swellby 234 ± 27%, mimicking genistein, whereas the inactive analog of PP2, PP3 (10 μM), had no effect. Moreover, block of PTP by orthovanadate (1 mM) inhibited ICl,swelland prevented its stimulation by PP2. In contrast with block of Src, block of EGFR kinase with PD-153035 (20 nM) inhibited ICl,swell. Several lines of evidence argue that the PP2-stimulated current was ICl,swell: 1) the stimulation was volume dependent, 2) the current was blocked by tamoxifen, 3) the current outwardly rectified with both symmetrical and physiological Cl−gradients, and 4) the current reversed near the Cl−equilibrium potential. To rule out contributions of other currents, Cd2+(0.2 mM) and Ba2+(1 mM) were added to the bath. Surprisingly, Cd2+suppressed the decay of ICl,swell, and Cd2+plus Ba2+eliminated time-dependent currents between −100 and +100 mV. Nevertheless, these divalent ions did not eliminate ICl,swellor prevent its stimulation by PP2. The results indicate that tyrosine phosphorylation controls ICl,swell, and regulation of ICl,swellby the Src and EGFR kinase families of PTK is antagonistic.