Differential effects of tyrosine kinase inhibitors on volume-sensitive chloride current in human atrial myocytes: evidence for dual regulation by Src and EGFR kinases

Abstract

To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (I(Cl.vol)) in human atrial myocytes and to identify the PTKs involved, we studied the effects of broad-spectrum and selective PTK inhibitors and the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (VO(4)(-3)). I(Cl.vol) evoked by hyposmotic bath solution (0.6-times isosmotic, 0.6T) was enhanced by genistein, a broad-spectrum PTK inhibitor, in a concentration-dependent manner (EC(50) = 22.4 microM); 100 microM genistein stimulated I(Cl.vol) by 122.4 +/- 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 150 microM) and tamoxifen (20 microM), blockers of I(Cl.vol). Moreover, the current augmented by genistein was volume dependent; it was abolished by hyperosmotic shrinkage in 1.4T, and genistein did not activate Cl(-) current in 1T. In contrast to the stimulatory effects of genistein, 100 microM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited I(Cl.vol) by 38.2 +/- 4.9% and 40.9 +/- 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter I(Cl.vol). In addition, the PTP inhibitor VO(4)(-3) (1 mM) reduced I(Cl.vol) by 53.5 +/- 4.5% (IC(50) = 249.6 microM). Pretreatment with VO(4)(-3) antagonized genistein-induced augmentation and A23- or A25-induced suppression of I(Cl.vol). Furthermore, the selective Src-family PTK inhibitor PP2 (5 microM) stimulated I(Cl.vol), mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 microM) reduced I(Cl.vol), mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO(4)(-3). The results suggest that I(Cl.vol) is regulated in part by the balance between PTK and PTP activity. Regulation is complex, however. Src and EGFR kinases, distinct soluble and receptor-mediated PTK families, have opposing effects on I(Cl.vol), and multiple target proteins are likely to be involved.

Figure 1.

I_Cl.vol in human atrial myocytes. (A) Time course of activation of current at +50 mV on switching from isosmotic (1T) to hyposmotic (0.6T) bath solution and full recovery in 1T. Currents at time points a–c shown at right. Currents were elicited by 300-ms steps to +50 from −40 mV (inset). (B) Voltage-dependent currents in 1T control (a), 0.6T (b), and 0.6T with 150 μM DIDS (c). DIDS, a blocker of I_Cl.vol, substantially inhibited the swelling-induced current. Arrows indicate 0 current. Voltage protocol for B and C, 300-ms steps to between −100 and +60 mV from −40 mV (inset). (C) Current-voltage (I-V) relationships for 0.6T-induced current (○) and 0.6T-induced current with 150 μM DIDS (•); difference currents were obtained by subtracting the current in 1T from that in 0.6T and 0.6T with DIDS. The 0.6T-induced current was outwardly rectifying and was significantly inhibited by DIDS at test potentials from −100 to −50 mV and from −20 to +60 mV (n = 6, P < 0.05 or P < 0.01). Block by DIDS was greater at positive than negative voltages, as expected for I_Cl.vol.

Figure 2.

Stimulation of I_Cl.vol by genistein. (A) Time course of current at +50 mV. I_Cl.vol was elicited by switching from 1T (a) to 0.6T (b) bath solution. Cells then were exposed to 100 μM genistein in 0.6T (c), and genistein was washed out (d). Genistein reversibly stimulated I_Cl.vol. Currents are shown at right. (B) Voltage-dependent current evoked in 1T control (a), 0.6T (b), and 0.6T with 100 μM genistein (c). Arrows indicate 0 current. (C) I-V relationships for I_Cl.vol (•) obtained by subtraction of currents before and after 0.6T exposure, with genistein exposure (▴), and drug washout (▵). *P < 0.05; **P < 0.01 vs. 0.6T. (D) Concentration-dependent stimulation of I_Cl.vol at +50 mV by genistein. EC₅₀ was 22.4 μM; number of cells at each concentration in parentheses. Voltage protocols are shown in insets.

Figure 3.

Lack of effect of daidzein on I_Cl.vol. (A) Time course of membrane current at +50 mV in 1T control (a), 0.6T (b), 0.6T with 100 μM daidzein (c), 100 μM genistein (d), and washout of genistein (e). After I_Cl.vol reached a steady-state in 0.6T, it was not significantly affected by daidzein but was reversibly stimulated by genistein. Protocol is shown in inset. (B) Currents at time points a–e in A. Arrow indicates 0 current. (C) I-V relationships for I_Cl.vol in absence (•) and presence (▵) of 100 μM daidzein (n = 5, P = NS).

Figure 4.

Tamoxifen blocks swelling- and genistein-induced current. (A) Time course of membrane current at +50 mV. I_Cl.vol activated upon switching from 1T to 0.6T and was augmented by 100 μM genistein. Tamoxifen (20 μM) substantially inhibited both the swelling- and genistein-induced currents. Currents at time points a–d at right. (B) Voltage-dependent currents in 1T control (a), 0.6T (b), 0.6T with 100 μM genistein (c), and 0.6T with genistein and 20 μM tamoxifen. Both the 0.6T- and genistein-stimulated currents were outwardly rectifying. Arrows indicate 0 current. Voltage protocols are shown in insets.

Figure 5.

Cell shrinkage reversed swelling- and genistein-induced activation of current. (A) Time course of membrane current at +50 mV. After I_Cl.vol was activated by switching from 1T (a) to 0.6T (b) and stimulated by 100 μM genistein (c), bathing solution was switched to hyperosmotic 1.4T with genistein. Currents at time points a–d are at right. (B) Voltage-dependent currents in 1T control (a), 0.6T (b), 0.6T with 100 μM genistein (c), and 1.4T with genistein (d). Arrows indicate 0 current. Voltage protocols are shown in insets.

Figure 6.

Inhibition of I_Cl.vol by VO₄ ⁻³. (A) Time course of current at +50 mV. I_Cl.vol was elicited by switching from 1T (a) to 0.6T (b) bath solution. Cells then were exposed to 1 mM VO₄ ⁻³ (OV) in 0.6T (c), and VO₄ ⁻³ was washed out (d). VO₄ ⁻³ reversibly inhibited I_Cl.vol. Currents are at right, and protocol is in inset. (B) I-V relationships for I_Cl.vol induced by 0.6T (•), with 1 mM VO₄ ⁻³ in 0.6T (▴), and after washout (▵). N = 6, *P < 0.05; **P < 0.01 vs. control I_Cl.vol. (C) Concentration-dependent inhibition of I_Cl.vol by VO₄ ⁻³. IC₅₀ was 249.6 μM; number of cells in parentheses.

Figure 7.

VO₄ ⁻³ (OV) antagonized the stimulation of I_Cl.vol by genistein. (A) Time course of current when order of exposure to genistein and VO₄ ⁻³ was reversed; 1T control (a), 0.6T (b), 1 mM VO₄ ⁻³ in 0.6T (c), and 100 μM geneistein plus VO₄ ⁻³ (d). Pretreatment with VO₄ ⁻³ significantly diminished stimulation of I_Cl.vol by genistein. Currents are at right. (B) Voltage-dependent I_Cl.vol obtained by digital subtraction of the current in 0.6T, after 1mM VO₄ ⁻³, and VO₄ ⁻³ with 100 μM genistein from that in 1T. (C) I-V relationships of I_Cl.vol obtained by digital subtraction of currents before and after the application of VO₄ ⁻³ (○), and VO₄ ⁻³ with 100 μM genistein (•). Although pretreatment with VO₄ ⁻³ significantly reduced stimulation of I_Cl.vol by genistein (compare with Fig. 2; P < 0.01), a small but significant stimulation was observed, 16.9 ± 2.5 and 18.9 ± 4.7% at −90 and +50 mV (P < 0.01, n = 7). Voltage protocols are shown in insets.

Figure 8.

Stimulation of I_Cl.vol by PP2. (A) Voltage-dependent I_Cl.vol was obtained by digital subtraction of the current in 0.6T, 5 μM PP2 for 10 min, and washout of PP2 for 30 min from that in 1T. Voltage protocols are shown in insets. (B) Voltage-dependent I_Cl.vol in 0.6T, after 1 mM VO₄ ⁻³ (OV) for 5 min, and VO₄ ⁻³ with 5 μM PP2 for additional 10 min C. Histograms summarize effects of 5 μM PP2, 1 mM VO₄ ⁻³, and VO₄ ⁻³ plus 5 μM PP2 on I_Cl.vol induced by swelling in 0.6T. Pretreatment with VO₄ ⁻³ almost completely antagonized the stimulation of I_Cl.vol by PP2.

Figure 9.

Inhibition of I_Cl.vol by tyrphostin A23. (A) Time course of current. I_Cl.vol was elicited by switching from 1T (a) to 0.6T (b) bath solution. Cells then were exposed to 100 μM A23 in 0.6T (c), and tyrphostin was washed out (d). A23 reversibly inhibited I_Cl.vol. Currents are at right, and protocol is in inset. (B) I-V relationships for I_Cl.vol induced by 0.6T (•), with 100 μM A23 (▴), and after washout of A23 (▵). *P < 0.05; **P < 0.01 vs. 0.6T-induced I_Cl.vol. (C) Concentration-dependent inhibition of I_Cl.vol by A23. IC₅₀ was 27.5 μM; number of cells in parentheses.

Figure 10.

Effect of tyrphostin A63 on I_Cl.vol. (A) Time course of current at +50 mV. I_Cl.vol was elicited by switching from 1T (a) to 0.6T (b) bath solution. Cells then were exposed to 100 μM A63 (c) and 100 μM A23 (d) in 0.6T. I_Cl.vol was not significantly affected by A63, an inactive analogue, but was blocked by A23 in the same cell. Protocol in inset. (B) Currents at time points a–d in A. (C) I-V relationships for I_Cl.vol in absence (•) and presence (▿) of 100 μM A63 (n = 5, P = NS).

Figure 11.

VO₄ ⁻³ (OV) antagonized the inhibition of I_Cl.vol by the tyrphostin A23. (A) Time course of current at +50 mV. I_Cl.vol was elicited by switching from 1T (a) to 0.6T (b) bath solution. Cells then were exposed to 1 mM VO₄ ⁻³ (OV, c) and VO₄ ⁻³ plus 100 μM A23 (d) in 0.6T. Pretreatment with VO₄ ⁻³ significantly diminished the block of I_Cl.vol by A23. Protocol is in inset. (B) Currents at time points a–d in A. (C) Histograms summarize effect of 100 μM A23, 1 mM VO₄ ⁻³, and VO₄ ⁻³ plus 100 μM A23 on I_Cl.vol induced by swelling in 0.6T. **P < 0.01 vs. 0.6T-induced I_Cl.vol; *P < 0.05 vs. 0.6T with VO₄ ⁻³; #P < 0.01 vs. 0.6T with A23.

Figure 12.

Suppression of I_Cl.vol by B56. (A) Voltage-dependent I_Cl.vol was obtained by digital subtraction of the current in 0.6T, 10 μM B56 for 10 min, and washout of B56 for 15 min from the current in 1T. (B) Voltage-dependent I_Cl.vol in 0.6T, after 1 mM VO₄ ⁻³ (OV) for 5 min, and VO₄ ⁻³ with 10 μM B56 for additional 10 min. Protocol in inset. (C) Histograms summarize effects of 10 μM B56, 1 mM VO₄ ⁻³, and VO₄ ⁻³ plus 10 μM B56 on I_Cl.vol at +50 mV induced by swelling in 0.6T (**P < 0.01 vs. 0.6T-induced I_Cl.vol). Pretreatment with VO₄ ⁻³ significantly antagonized the inhibition of I_Cl.vol by B56 (**P < 0.01 vs. B56, #P<0.05 vs. VO₄ ⁻³).

Figure 13.

Schematic model of biphasic regulation of I_Cl.vol by Src family and EGFR PTKs and PTPs. Two tyrosine phosphorylation sites, Tyr 1 and Tyr 2, on the I_Cl.vol channel or signaling molecules are postulated. Tyr1 is phosphorylated by a PP2- and genistein-sensitive Src family kinase and dephosphorylated by a VO₄ ⁻³-sensitive PTP (PTP_a). Phosphorylation of Tyr1 by one or more steps (dashed line) favors I_Cl.vol channel closure, whereas dephosphorylation favors opening. Tyr2 is phosphorylated by tyrphostin-sensitive EGFR kinase and dephosphorylated by a VO₄ ⁻³-sensitive PTP (PTP_b). In contrast to Tyr1, phosphorylation of Tyr2 favors channel opening, whereas dephosphorylation favors closure. The effect of VO₄ ⁻³ on Tyr1 (thick lines) was dominant over that on Tyr2 (thin lines). On the other hand, genistein did not activate I_Cl.vol in 1T or maintain its activation after osmotic shrinkage (thick solid lines). Therefore, tyrosine phosphorylation appears to modulate channel activity rather than directly control gating.