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Immunomodulation of voltage-dependent K+ channels in macrophages: molecular and biophysical consequences.

Villalonga N, David M, Bielanska J, Vicente R, Comes N, Valenzuela C, Felipe A - J. Gen. Physiol. (2010)

Bottom Line: An increase in K(+) current amplitude in lipopolysaccharide-activated macrophages is characterized by a faster C-type inactivation, a greater percentage of cumulative inactivation, and a more effective margatoxin (MgTx) inhibition than control cells.In contrast, dexamethasone decreased the C-type inactivation, the cumulative inactivation, and the sensitivity to MgTx concomitantly with a decrease in K(v)1.3 expression.Our results demonstrate that the immunomodulation of macrophages triggers molecular and biophysical consequences in K(v)1.3/K(v)1.5 hybrid channels by altering the subunit stoichiometry.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Physiology Laboratory, Departament de Bioquímica i Biología Molecular, Institut de Biomedicina, Universitat de Barcelona, E-08028 Barcelona, Spain.

ABSTRACT
Voltage-dependent potassium (K(v)) channels play a pivotal role in the modulation of macrophage physiology. Macrophages are professional antigen-presenting cells and produce inflammatory and immunoactive substances that modulate the immune response. Blockage of K(v) channels by specific antagonists decreases macrophage cytokine production and inhibits proliferation. Numerous pharmacological agents exert their effects on specific target cells by modifying the activity of their plasma membrane ion channels. Investigation of the mechanisms involved in the regulation of potassium ion conduction is, therefore, essential to the understanding of potassium channel functions in the immune response to infection and inflammation. Here, we demonstrate that the biophysical properties of voltage-dependent K(+) currents are modified upon activation or immunosuppression in macrophages. This regulation is in accordance with changes in the molecular characteristics of the heterotetrameric K(v)1.3/K(v)1.5 channels, which generate the main K(v) in macrophages. An increase in K(+) current amplitude in lipopolysaccharide-activated macrophages is characterized by a faster C-type inactivation, a greater percentage of cumulative inactivation, and a more effective margatoxin (MgTx) inhibition than control cells. These biophysical parameters are related to an increase in K(v)1.3 subunits in the K(v)1.3/K(v)1.5 hybrid channel. In contrast, dexamethasone decreased the C-type inactivation, the cumulative inactivation, and the sensitivity to MgTx concomitantly with a decrease in K(v)1.3 expression. Neither of these treatments apparently altered the expression of K(v)1.5. Our results demonstrate that the immunomodulation of macrophages triggers molecular and biophysical consequences in K(v)1.3/K(v)1.5 hybrid channels by altering the subunit stoichiometry.

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Macrophages express Kv1.3 and Kv1.5. Cells were held at −80 mV, and pulse potentials were applied as indicated. (A) Representative traces of delayed rectifier K+ currents. (B) Steady-state activation curve of the outward current. Conductance was plotted against test potentials. (C) mRNA expression of Kv1.3 and Kv1.5 in Raw 264.7 cells. Mouse brain and heart RNA were used as positive controls for Kv1.3 and Kv1.5, respectively. PCR reactions were performed in the presence (+) or absence (−) of the retrotranscriptase reaction. (D) Kv1.3 and Kv1.5 protein expression in Raw macrophages. Jurkat T lymphocytes and L6E9 skeletal muscle myoblasts were used as positive controls for Kv1.3 and Kv1.5, respectively. (E) Immunocytochemical electron microscopic detection of Kv1.3 and Kv1.5 proteins. Arrows show specific channel protein localization. Black arrow, Kv1.3; white arrow, Kv1.5; bar, 0.20 µm.
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fig1: Macrophages express Kv1.3 and Kv1.5. Cells were held at −80 mV, and pulse potentials were applied as indicated. (A) Representative traces of delayed rectifier K+ currents. (B) Steady-state activation curve of the outward current. Conductance was plotted against test potentials. (C) mRNA expression of Kv1.3 and Kv1.5 in Raw 264.7 cells. Mouse brain and heart RNA were used as positive controls for Kv1.3 and Kv1.5, respectively. PCR reactions were performed in the presence (+) or absence (−) of the retrotranscriptase reaction. (D) Kv1.3 and Kv1.5 protein expression in Raw macrophages. Jurkat T lymphocytes and L6E9 skeletal muscle myoblasts were used as positive controls for Kv1.3 and Kv1.5, respectively. (E) Immunocytochemical electron microscopic detection of Kv1.3 and Kv1.5 proteins. Arrows show specific channel protein localization. Black arrow, Kv1.3; white arrow, Kv1.5; bar, 0.20 µm.

Mentions: Kv currents were evoked in Raw cells by applying depolarizing pulses from a holding potential of −80 mV to different depolarizing voltages from −80 to +60 mV in 10-mV steps (Fig. 1 A). Fig. 1 B plots normalized conductance against test potential. The threshold for activation was about −20 mV. Vh and k slope were 11.1 ± 2.2 and 12.3 ± 2.5 mV, respectively. Previous work indicates that macrophages exhibit Kv currents mainly generated by Kv1.3 and Kv1.5 channels (Vicente et al., 2003, 2005, 2006, 2008; Villalonga et al., 2007). Therefore, we analyzed the presence of these channels in Raw macrophages. To analyze the K+ channel mRNA expression, we performed RT-PCR analysis. Mouse brain and heart RNAs were used as positive controls for Kv1.3 and Kv1.5, respectively. Fig. 1 C demonstrates that Kv1.3 and Kv1.5 mRNA were detected in Raw macrophages in the presence (+RT), but not in the absence, of the retrotranscriptase (−RT). In addition, a specific Kv1.3 and Kv1.5 signal was obtained by Western blot (Fig. 1 D). As expected, Kv1.3 protein was detected in macrophages and Jurkat T cells, whereas Raw cells shared Kv1.5 expression with L6E9 skeletal muscle myoblasts (Villalonga et al., 2008). We have recently shown that although Kv1.3 may form homotetrameric structures, and may be located at the membrane surface, Kv1.5 mostly coassociates with Kv1.3 to form heterotetrameric channels in macrophages (Vicente et al., 2006; Villalonga et al., 2007). In addition, the presence of Kv1.5 impairs the membrane surface location of the complex (Martínez-Mármol et al., 2008; Vicente et al., 2008). Electron microscopic immunocytochemical detection studies with specific antibodies showed that although Kv1.3 was detected at the membrane surface, many heteromeric structures containing Kv1.5 were located intracellularly (Fig. 1 E). Collectively, these data indicate that Raw 264.7 macrophages have outward delayed rectifier K+ currents that are mainly conducted by Kv1.3 and Kv1.5 channels.


Immunomodulation of voltage-dependent K+ channels in macrophages: molecular and biophysical consequences.

Villalonga N, David M, Bielanska J, Vicente R, Comes N, Valenzuela C, Felipe A - J. Gen. Physiol. (2010)

Macrophages express Kv1.3 and Kv1.5. Cells were held at −80 mV, and pulse potentials were applied as indicated. (A) Representative traces of delayed rectifier K+ currents. (B) Steady-state activation curve of the outward current. Conductance was plotted against test potentials. (C) mRNA expression of Kv1.3 and Kv1.5 in Raw 264.7 cells. Mouse brain and heart RNA were used as positive controls for Kv1.3 and Kv1.5, respectively. PCR reactions were performed in the presence (+) or absence (−) of the retrotranscriptase reaction. (D) Kv1.3 and Kv1.5 protein expression in Raw macrophages. Jurkat T lymphocytes and L6E9 skeletal muscle myoblasts were used as positive controls for Kv1.3 and Kv1.5, respectively. (E) Immunocytochemical electron microscopic detection of Kv1.3 and Kv1.5 proteins. Arrows show specific channel protein localization. Black arrow, Kv1.3; white arrow, Kv1.5; bar, 0.20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812499&req=5

fig1: Macrophages express Kv1.3 and Kv1.5. Cells were held at −80 mV, and pulse potentials were applied as indicated. (A) Representative traces of delayed rectifier K+ currents. (B) Steady-state activation curve of the outward current. Conductance was plotted against test potentials. (C) mRNA expression of Kv1.3 and Kv1.5 in Raw 264.7 cells. Mouse brain and heart RNA were used as positive controls for Kv1.3 and Kv1.5, respectively. PCR reactions were performed in the presence (+) or absence (−) of the retrotranscriptase reaction. (D) Kv1.3 and Kv1.5 protein expression in Raw macrophages. Jurkat T lymphocytes and L6E9 skeletal muscle myoblasts were used as positive controls for Kv1.3 and Kv1.5, respectively. (E) Immunocytochemical electron microscopic detection of Kv1.3 and Kv1.5 proteins. Arrows show specific channel protein localization. Black arrow, Kv1.3; white arrow, Kv1.5; bar, 0.20 µm.
Mentions: Kv currents were evoked in Raw cells by applying depolarizing pulses from a holding potential of −80 mV to different depolarizing voltages from −80 to +60 mV in 10-mV steps (Fig. 1 A). Fig. 1 B plots normalized conductance against test potential. The threshold for activation was about −20 mV. Vh and k slope were 11.1 ± 2.2 and 12.3 ± 2.5 mV, respectively. Previous work indicates that macrophages exhibit Kv currents mainly generated by Kv1.3 and Kv1.5 channels (Vicente et al., 2003, 2005, 2006, 2008; Villalonga et al., 2007). Therefore, we analyzed the presence of these channels in Raw macrophages. To analyze the K+ channel mRNA expression, we performed RT-PCR analysis. Mouse brain and heart RNAs were used as positive controls for Kv1.3 and Kv1.5, respectively. Fig. 1 C demonstrates that Kv1.3 and Kv1.5 mRNA were detected in Raw macrophages in the presence (+RT), but not in the absence, of the retrotranscriptase (−RT). In addition, a specific Kv1.3 and Kv1.5 signal was obtained by Western blot (Fig. 1 D). As expected, Kv1.3 protein was detected in macrophages and Jurkat T cells, whereas Raw cells shared Kv1.5 expression with L6E9 skeletal muscle myoblasts (Villalonga et al., 2008). We have recently shown that although Kv1.3 may form homotetrameric structures, and may be located at the membrane surface, Kv1.5 mostly coassociates with Kv1.3 to form heterotetrameric channels in macrophages (Vicente et al., 2006; Villalonga et al., 2007). In addition, the presence of Kv1.5 impairs the membrane surface location of the complex (Martínez-Mármol et al., 2008; Vicente et al., 2008). Electron microscopic immunocytochemical detection studies with specific antibodies showed that although Kv1.3 was detected at the membrane surface, many heteromeric structures containing Kv1.5 were located intracellularly (Fig. 1 E). Collectively, these data indicate that Raw 264.7 macrophages have outward delayed rectifier K+ currents that are mainly conducted by Kv1.3 and Kv1.5 channels.

Bottom Line: An increase in K(+) current amplitude in lipopolysaccharide-activated macrophages is characterized by a faster C-type inactivation, a greater percentage of cumulative inactivation, and a more effective margatoxin (MgTx) inhibition than control cells.In contrast, dexamethasone decreased the C-type inactivation, the cumulative inactivation, and the sensitivity to MgTx concomitantly with a decrease in K(v)1.3 expression.Our results demonstrate that the immunomodulation of macrophages triggers molecular and biophysical consequences in K(v)1.3/K(v)1.5 hybrid channels by altering the subunit stoichiometry.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Physiology Laboratory, Departament de Bioquímica i Biología Molecular, Institut de Biomedicina, Universitat de Barcelona, E-08028 Barcelona, Spain.

ABSTRACT
Voltage-dependent potassium (K(v)) channels play a pivotal role in the modulation of macrophage physiology. Macrophages are professional antigen-presenting cells and produce inflammatory and immunoactive substances that modulate the immune response. Blockage of K(v) channels by specific antagonists decreases macrophage cytokine production and inhibits proliferation. Numerous pharmacological agents exert their effects on specific target cells by modifying the activity of their plasma membrane ion channels. Investigation of the mechanisms involved in the regulation of potassium ion conduction is, therefore, essential to the understanding of potassium channel functions in the immune response to infection and inflammation. Here, we demonstrate that the biophysical properties of voltage-dependent K(+) currents are modified upon activation or immunosuppression in macrophages. This regulation is in accordance with changes in the molecular characteristics of the heterotetrameric K(v)1.3/K(v)1.5 channels, which generate the main K(v) in macrophages. An increase in K(+) current amplitude in lipopolysaccharide-activated macrophages is characterized by a faster C-type inactivation, a greater percentage of cumulative inactivation, and a more effective margatoxin (MgTx) inhibition than control cells. These biophysical parameters are related to an increase in K(v)1.3 subunits in the K(v)1.3/K(v)1.5 hybrid channel. In contrast, dexamethasone decreased the C-type inactivation, the cumulative inactivation, and the sensitivity to MgTx concomitantly with a decrease in K(v)1.3 expression. Neither of these treatments apparently altered the expression of K(v)1.5. Our results demonstrate that the immunomodulation of macrophages triggers molecular and biophysical consequences in K(v)1.3/K(v)1.5 hybrid channels by altering the subunit stoichiometry.

Show MeSH
Related in: MedlinePlus