UVM Theses and Dissertations
Format:
Print
Author:
Dey, Deblina
Dept./Program:
Biology
Year:
2010
Degree:
MS
Abstract:
Voltage-activated Ca² channels are an important route of Ca²⁺ entry in excitable cells. Intracellular Ca²⁺ accumulation evoked by the opening of voltage-activated Ca²⁺ channels regulates a variety of cellular events including neurotransmitter release, cell proliferation and differentiation. Based on their biophysical and pharmacological properties, voltage activated Ca²⁺ channels are broadly divided into low-voltage and high voltage-activated Ca²⁺ channels (LVA and HVA, respectively). LVA Ca²⁺ channels generate transient currents at relatively hyperpolarized potentials for what they are also known as T-type Ca²⁺ channels. Although the physiological role of T-type Ca²⁺ channels is well established, we have very little understanding about the cellular and molecular events involved in the regulation of T-type Ca²⁺ channel expression.
Previous studies have demonstrated that neuropoietic cytokines such as Ciliary Neurotrophic Factor (CNTF) or Leukemia Inhibitory Factor (LIF) stimulate the functional expression of T-type Ca² channels in nodose sensory neurons in the chicken embryo. CNTF-induced channel expression requires 12 hr stimulation in order to reach maximal expression and is not affected by inhibition of protein synthesis, suggesting the involvement of a post-translational mechanism. Disruption of the Golgi apparatus with brefeldin-A inhibits the stimulatory effect of CNTF, suggesting that protein trafficking regulates the functional expression of T-type Ca²⁺ channels.
These results have helped to formulate our hypothesis that neuropoietic cytokines regulate the functional expression of Cav3.2 T-type Ca² channels by stimulating trafficking of channel proteins to the membrane. To test this hypothesis, we used human embryonic kidney (HEK 293) cells transfected with a green fluorescent protein (GFP)-tagged Cav3.2 rat [alpha]1H subunit. Functional expression of Cav3.2 T-type Ca²⁺ channels was assesses following overnight stimulation with the neuropoietic cytokine LIF. HEK 293 cells transfected with GFP-tagged Cav3.2 [alpha]1H subunits expressed a basal Ca²⁺ current. Overnight treatment of [alpha]1H-GFP transfected cells with LIF caused a significant increase in the functional expression of T-type Ca²⁺ channels as indicated by changes in current density. LIF also evoked a significant increase in the GFP membrane fluorescence when compared with untreated cells. Disruption ofthe Golgi apparatus with brefeldin-A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca²⁺ channels.
Trafficking of [alpha]1H-GFP was also disrupted by co-transfection of HEK 293 cells with the dominant negative form of ADP ribosylation factor (ARF) ARF1 but not ARF6, suggesting that the ADP-ribosylation factor ARF1regulates the LIF-evoked membrane trafficking of [alpha]1H-GFP subunits. Trafficking of T-type Ca² channels required transient activation of the Janus activated kinases or JAK and mitogen activated protein kinases or ERK signaling pathways since stimulation of HEK 293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pre-treatment of HEK 293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca²⁺ channel expression. These findings demonstrate that cytokines such as LIF promote the trafficking of T-type Ca²⁺ channels.
Previous studies have demonstrated that neuropoietic cytokines such as Ciliary Neurotrophic Factor (CNTF) or Leukemia Inhibitory Factor (LIF) stimulate the functional expression of T-type Ca² channels in nodose sensory neurons in the chicken embryo. CNTF-induced channel expression requires 12 hr stimulation in order to reach maximal expression and is not affected by inhibition of protein synthesis, suggesting the involvement of a post-translational mechanism. Disruption of the Golgi apparatus with brefeldin-A inhibits the stimulatory effect of CNTF, suggesting that protein trafficking regulates the functional expression of T-type Ca²⁺ channels.
These results have helped to formulate our hypothesis that neuropoietic cytokines regulate the functional expression of Cav3.2 T-type Ca² channels by stimulating trafficking of channel proteins to the membrane. To test this hypothesis, we used human embryonic kidney (HEK 293) cells transfected with a green fluorescent protein (GFP)-tagged Cav3.2 rat [alpha]1H subunit. Functional expression of Cav3.2 T-type Ca²⁺ channels was assesses following overnight stimulation with the neuropoietic cytokine LIF. HEK 293 cells transfected with GFP-tagged Cav3.2 [alpha]1H subunits expressed a basal Ca²⁺ current. Overnight treatment of [alpha]1H-GFP transfected cells with LIF caused a significant increase in the functional expression of T-type Ca²⁺ channels as indicated by changes in current density. LIF also evoked a significant increase in the GFP membrane fluorescence when compared with untreated cells. Disruption ofthe Golgi apparatus with brefeldin-A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca²⁺ channels.
Trafficking of [alpha]1H-GFP was also disrupted by co-transfection of HEK 293 cells with the dominant negative form of ADP ribosylation factor (ARF) ARF1 but not ARF6, suggesting that the ADP-ribosylation factor ARF1regulates the LIF-evoked membrane trafficking of [alpha]1H-GFP subunits. Trafficking of T-type Ca² channels required transient activation of the Janus activated kinases or JAK and mitogen activated protein kinases or ERK signaling pathways since stimulation of HEK 293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pre-treatment of HEK 293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca²⁺ channel expression. These findings demonstrate that cytokines such as LIF promote the trafficking of T-type Ca²⁺ channels.