Antagonistic regulation of swelling-activated Cl- current in rabbit ventricle by Src and EGFR protein tyrosine kinases - PubMed (original) (raw)

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

Zuojun Ren et al. Am J Physiol Heart Circ Physiol. 2005 Jun.

Abstract

Regulation of swelling-activated Cl(-) current (I(Cl,swell)) is complex, and multiple signaling cascades are implicated. To determine whether protein tyrosine kinase (PTK) modulates I(Cl,swell) and 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). I(Cl,swell) evoked by hyposmotic swelling was increased 181 +/- 17% by 100 microM genistein, and the genistein-induced current was blocked by the selective I(Cl,swell) blocker tamoxifen (10 microM). Block of Src with PP2 (10 microM) stimulated tamoxifen-sensitive I(Cl,swell) by 234 +/- 27%, mimicking genistein, whereas the inactive analog of PP2, PP3 (10 microM), had no effect. Moreover, block of PTP by orthovanadate (1 mM) inhibited I(Cl,swell) and prevented its stimulation by PP2. In contrast with block of Src, block of EGFR kinase with PD-153035 (20 nM) inhibited I(Cl,swell). Several lines of evidence argue that the PP2-stimulated current was I(Cl,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, Cd(2+) (0.2 mM) and Ba(2+) (1 mM) were added to the bath. Surprisingly, Cd(2+) suppressed the decay of I(Cl,swell), and Cd(2+) plus Ba(2+) eliminated time-dependent currents between -100 and +100 mV. Nevertheless, these divalent ions did not eliminate I(Cl,swell) or prevent its stimulation by PP2. The results indicate that tyrosine phosphorylation controls I(Cl,swell), and regulation of I(Cl,swell) by the Src and EGFR kinase families of PTK is antagonistic.

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Figures

Fig. 1

Fig. 1

Genistein, a broad-spectrum protein tyrosine kinase (PTK) blocker, augmented swelling-activated Cl− current (_I_Cl,swell). A: current-voltage (I-V) relationship in isosmotic (1T) solution, after swelling in hypoosmotic (0.7T) solution for 10 min, after exposure to 100 μM genistein in 0.7T solution for 10 min (Gen), and after addition of 10 μM tamoxifen (Tam), a selective _I_Cl,swell blocker, to 0.7T plus genistein solution. I-V curves cross near the Cl− equilibrium potential (_E_Cl), which is −42 mV. B: families of swelling-induced difference currents. _I_Cl,swell partially inactivated at positive potentials. C: genistein-induced difference currents in 0.7T solution. D: tamoxifen-sensitive difference currents in 0.7T plus genistein solution. Swelling activated outwardly rectifying _I_Cl,swell that reversed near _E_Cl and was stimulated by genistein. Tamoxifen blocked both the swelling- and genistein-induced components. Calibrations apply to all current records.

Fig. 2

Fig. 2

_I_Cl,swell is stimulated by PP2, a selective inhibitor of Src family PTKs, but not by its inactive analog, PP3. A: I-V relationships in 1T solution, after swelling in 0.7T solution for 10 min, after exposure to 10 μM PP2 in 0.7T solution for 10 min (PP2), and after addition of 10 μM tamoxifen to 0.7T solution plus PP2. B: families of swelling-induced difference currents. C: PP2-induced difference currents in 0.7T solution. D: tamoxifen-sensitive difference currents in 0.7T plus PP2 solution. Both the PP2- and swelling-induced currents were sensitive to 10 μM tamoxifen. Selective inhibition of Src by PP2 mimicked the effect of genistein. In contrast, PP3 (an inactive analog of PP2; 10 μM for 10 min) did not alter the magnitude or time course of membrane currents in 0.7T solution. E: PP3-induced difference currents in 0.7T solution. These records were obtained in a different cell than for _A_-D.

Fig. 3

Fig. 3

Stimulation of _I_Cl,swell by PP2 requires cell swelling. I-V relationships in 1T and after exposure to 10 μM PP2 in 1T solution for 10 min are shown. Inset: families of PP2-induced difference currents in 1T solution. PP2 did not alter the outwardly rectifying background Cl− current attributed at least in part to _I_Cl,swell under isosmotic conditions. Note the change in scale of I-V relationship compared with previous figures.

Fig. 4

Fig. 4

Cd2+ (0.2 mM) suppressed time dependence of _I_Cl,swell at positive potentials and revealed a delayed rectifier current. Examples from two myocytes are shown. A, B, C: data from one myocyte. D, E, F: data from a second myocyte. Families of currents after swelling in 0.7T solution (A and D), after exposure to Cd2+ (B and E), and Cd2+ -sensitive current (C and F) are shown. Cd2+ inhibited the time dependence of _I_Cl,swell at positive potentials and had a variable effect on the steady-state current. After Cd2+ block, a slowly activating outward component of current was apparent. Block of _I_Cl,swell was greatest in the second myocyte (_D_-F).

Fig. 5

Fig. 5

Cd2+ plus Ba2+ eliminated the time dependence of membrane currents but not the stimulation of _I_Cl,swell by PP2. A: I-V relationships in the presence of 0.2 mM Cd2+ and 1 mM Ba2+ in 1T solution, after swelling in 0.7T solution for 10 min, after exposure to 10 μM PP2 for 10 min in 0.7T solution, and after addition of 10 μM tamoxifen for 10 min to block _I_Cl,swell. B, C, D, and E: families of currents in 1T solution, 0.7T solution, 0.7T solution plus PP2, and 0.7T solution plus PP2 and tamoxifen, respectively. PP2 stimulated an outwardly rectifying, tamoxifen-sensitive current that reversed near _E_Cl.

Fig. 6

Fig. 6

Cd2+ plus Ba2+ eliminated the time dependence of _I_Cl,swell at strongly positive potentials. To test whether Cd2+ (0.2 mM) and Ba2+ (1 mM) shifted the onset of _I_Cl,swell inactivation to more positive voltages, the membrane voltage was stepped from −60 mV to potentials between −60 and +100 mV. A: I-V relationships in 1T solution and after 10 min of swelling in 0.7T solution. B and C: families of currents in 1T and 0.7T solutions, respectively. D: swelling-induced difference current. _I_Cl,swell remained time independent to at least +100 mV.

Fig. 7

Fig. 7

Cl− currents in symmetrical high- Cl− solutions (intracellular and extracellular Cl− concentrations = 98.6 mM). A: I-V relationships for currents in 1T solution, after 10 min of swelling in 0.7T solution, and after 10 min of exposure to 10 μM PP2 in 0.7T solution. B, C, and D: families of currents in 1T, 0.7T, and 0.7T plus PP2 solution, respectively. Swelling and PP2 stimulated outwardly rectifying, time-independent currents that reversed near 0 mV in symmetrical Cl− solutions with 0.2 mM Cd2+ and 1 mM Ba2+ in the bath.

Fig. 8

Fig. 8

Block of protein tyrosine phosphatase (PTP) by orthovanadate partially inhibits _I_Cl,swell and prevents stimulation of _I_Cl,swell upon block of Src by PP2. A: I-V relationships for currents in 1T solution, after 10 min of swelling in 0.7T solution, after exposure to 1 mM orthovanadate in 0.7T solution for 10 min (OV), and after addition of 10 μM PP2 for 10 min. B, C, D, and E: families of currents in 1T, 0.7T, 0.7T plus orthovanadate, and 0.7T plus orthovanadate and PP2 solution, respectively.

Fig. 9

Fig. 9

Block of epidermal growth factor receptor (EGFR) kinase by PD-153035 inhibits _I_Cl,swell. A: I-V relationships for currents in 1T solution, after 10 min of swelling in 0.7T solution, and after exposure to 20 nM PD-153035 in 0.7T solution for 12–15 min. B, C, and D: families of currents in 1T, 0.7T, and 0.7T plus PD-153035 solution, respectively.

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References

    1. Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem. 1987;262:5592–5595. - PubMed
    1. Bahinski A, Nairn AC, Greengard P, Gadsby DC. Chloride conductance regulated by cyclic AMP-dependent protein kinase in cardiac myocytes. Nature. 1989;340:718–721. - PubMed
    1. Bain J, McLauchlan H, Elliott M, Cohen P. The specificities of protein kinase inhibitors: an update. Biochem J. 2003;371:199–204. - PMC - PubMed
    1. Baumgarten CM, Clemo HF. Swelling-activated chloride channels in cardiac physiology and pathophysiology. Prog Biophys Mol Biol. 2003;82:25–42. - PubMed
    1. Bond TD, Ambikapathy S, Mohammad S, Valverde MA. Osmo-sensitive Cl− currents and their relevance to regulatory volume decrease in human intestinal T84 cells: outwardly vs. inwardly rectifying currents. J Physiol (Lond) 1998;511:45–54. - PMC - PubMed

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