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Published on: October 27, 2010
Regulation of swelling-activated chloride channels in embryonic chick heart cells
Hua Wei1, Yan Ai Mei, Jia Ting Sun
1Department of Physiology and Biophysics, Liren Laboratory, School of Life Sciences, Fudan University, Shanghai 200433, China.
Abstract:
Swelling-activated Cl- currents, I(Cl,swell) were measured during hyposmotic shock in white Leghorn embryonic chick heart cells using the whole-cell recording of patch-clamp technique. Genistein, an inhibitor of protein tyrosine kinase (PTK), suppressed I(Cl,swell). Under isosmotic condition phorbol 12-myristate 13-acetate (PMA), an activator of PKC, elicited the Cl- current similar to that in hyposmotic solution, whereas hyposmotic shock did not elicit I(Cl,swell) in chelerythrine chloride(an inhibitor of PKC)-treated cells. Confocal microscopy experiments using FITC-phalloidin as a fluorescent label of F-actin showed that the actin network was moved from cortical region of the cell to the center after hyposmotic shock as compared with the image under isosmotic condition. When the cells were treated with cytochalasin B (CB) or cytochalasin D (CD) under isosmotic condition the disruption of the F-actin integrity was observed, and I(Cl,swell) was not elicited. With combination treatment of CB with PMA, hyposmotic solution could not elicited I(Cl,swell). The results suggested that the role of PTK, probably receptor tyrosine kinase, for regulation of I(Cl,swell) appeared to be at upstream site related to the role of F-actin. Then PKC signal pathway was activated somehow and finally change in the polymerization state of cytoskeleton led to activate the swelling-activated Cl- channels. These results demonstrate clearly that PTK, PKC and F-actin are important factors for regulation of I(Cl,swell), in embryonic chick heart cells as compared with often controversial results reported in different cell types.
Insights
Swelling-activated chloride currents in embryonic chick heart cells are regulated by protein tyrosine kinase (PTK) and protein kinase C (PKC) signaling pathways, involving changes in the actin cytoskeleton. These factors are crucial for activating swelling-activated chloride channels.
Area of Science:
- Cellular Physiology
- Molecular Biology
- Biophysics
Background:
- Swelling-activated chloride currents (I(Cl,swell)) play roles in cell volume regulation.
- The precise molecular mechanisms controlling I(Cl,swell) activation remain incompletely understood across different cell types.
- Embryonic chick heart cells offer a model system to investigate these mechanisms.
Purpose of the Study:
- To elucidate the roles of protein tyrosine kinase (PTK), protein kinase C (PKC), and the actin cytoskeleton in regulating swelling-activated chloride currents in embryonic chick heart cells.
- To determine the upstream and downstream signaling events involved in I(Cl,swell) activation.
Main Methods:
- Whole-cell patch-clamp recordings were used to measure I(Cl,swell) during hyposmotic shock.
- Pharmacological inhibitors (genistein, chelerythrine chloride) and activators (phorbol 12-myristate 13-acetate) of signaling pathways were employed.
- Confocal microscopy with FITC-phalloidin was used to visualize F-actin organization.
- Cytochalasin B and D were used to disrupt the actin cytoskeleton.
Main Results:
- Genistein (PTK inhibitor) suppressed I(Cl,swell).
- Phorbol 12-myristate 13-acetate (PKC activator) mimicked hyposmotic shock effects, while chelerythrine chloride (PKC inhibitor) blocked I(Cl,swell) activation by hyposmotic shock.
- Hyposmotic shock induced a shift in F-actin from the cell periphery to the center.
- Disruption of F-actin integrity with cytochalasin B/D prevented I(Cl,swell) activation, even with concurrent PMA treatment.
Conclusions:
- PTK signaling, likely upstream, influences I(Cl,swell) regulation.
- PKC pathway activation is involved in the signaling cascade leading to channel activation.
- Changes in F-actin polymerization state are critical for activating swelling-activated chloride channels.
- PTK, PKC, and F-actin are essential regulators of I(Cl,swell) in embryonic chick heart cells.
