Abstract
We report here the expression and properties of the intermediate- conductance Ca2+-activated K+ (IKCa) channel in the GL-15 human glioblastoma cell line. Macroscopic IKCa currents on GL-15 cells displayed a mean amplitude of 7.2±0.8 pA/pF at 0 mV, at day 1 after plating. The current was inhibited by clotrimazole (CTL, IC 50=257 nM), TRAM-34 (IC50=55 nM), and charybdotoxin (CTX, IC50=10.3 nM). RT-PCR analysis demonstrated the expression of mRNA encoding the IKCa channel in GL-15 cells. Unitary currents recorded using the inside-out configuration had a conductance of 25 pS, a KD for Ca2+ of 188 nM at -100 mV, and no voltage dependence. We tested whether the IKCa channel expression in GL-15 cells could be the result of an increased ERK activity. Inhibition of the ERK pathway with the MEK antagonist PD98059 (25 μM, for 5 days) virtually suppressed the IK Ca current in GL-15 cells. PD98059 treatment also increased the length of cellular processes and up-regulated the astrocytic differentiative marker GFAP. A significant reduction of the IKCa current amplitude was also observed with time in culture, with mean currents of 7.17±0.75 pA/pF at 1-2 days, and 3.11±1.35 pA/pF at 5-6 days after plating. This time-dependent down-regulation of the IKCa current was not accompanied by changes in the ERK activity, as assessed by immunoblot analysis. Semiquantitative RT-PCR analysis demonstrated a ∼35% reduction of the IKCa channel mRNA resulting from ERK inhibition and a ∼50% reduction with time in culture.
| Original language | English |
|---|---|
| Pages (from-to) | 47-56 |
| Number of pages | 10 |
| Journal | Cellular Physiology and Biochemistry |
| Volume | 18 |
| Issue number | 1-3 |
| Publication status | Published - 2006 |
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Keywords
- Cell differentiation
- ERK
- GFAP
- Human glioblastoma GL-15 cell line
- Intermediate-conductance Ca-activated K channels
ASJC Scopus subject areas
- Physiology
- Cell Biology
Cite this
Expression and modulation of the intermediate-conductance Ca 2+-activated K+ channel in glioblastoma GL-15 cells. / Fioretti, Bernard; Castigli, Emilia; Micheli, Maria R.; Bova, Rodolfo; Sciaccaluga, Miriam; Harper, Alexander; Franciolini, Fabio; Catacuzzeno, Luigi.
In: Cellular Physiology and Biochemistry, Vol. 18, No. 1-3, 2006, p. 47-56.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Expression and modulation of the intermediate-conductance Ca 2+-activated K+ channel in glioblastoma GL-15 cells
AU - Fioretti, Bernard
AU - Castigli, Emilia
AU - Micheli, Maria R.
AU - Bova, Rodolfo
AU - Sciaccaluga, Miriam
AU - Harper, Alexander
AU - Franciolini, Fabio
AU - Catacuzzeno, Luigi
PY - 2006
Y1 - 2006
N2 - We report here the expression and properties of the intermediate- conductance Ca2+-activated K+ (IKCa) channel in the GL-15 human glioblastoma cell line. Macroscopic IKCa currents on GL-15 cells displayed a mean amplitude of 7.2±0.8 pA/pF at 0 mV, at day 1 after plating. The current was inhibited by clotrimazole (CTL, IC 50=257 nM), TRAM-34 (IC50=55 nM), and charybdotoxin (CTX, IC50=10.3 nM). RT-PCR analysis demonstrated the expression of mRNA encoding the IKCa channel in GL-15 cells. Unitary currents recorded using the inside-out configuration had a conductance of 25 pS, a KD for Ca2+ of 188 nM at -100 mV, and no voltage dependence. We tested whether the IKCa channel expression in GL-15 cells could be the result of an increased ERK activity. Inhibition of the ERK pathway with the MEK antagonist PD98059 (25 μM, for 5 days) virtually suppressed the IK Ca current in GL-15 cells. PD98059 treatment also increased the length of cellular processes and up-regulated the astrocytic differentiative marker GFAP. A significant reduction of the IKCa current amplitude was also observed with time in culture, with mean currents of 7.17±0.75 pA/pF at 1-2 days, and 3.11±1.35 pA/pF at 5-6 days after plating. This time-dependent down-regulation of the IKCa current was not accompanied by changes in the ERK activity, as assessed by immunoblot analysis. Semiquantitative RT-PCR analysis demonstrated a ∼35% reduction of the IKCa channel mRNA resulting from ERK inhibition and a ∼50% reduction with time in culture.
AB - We report here the expression and properties of the intermediate- conductance Ca2+-activated K+ (IKCa) channel in the GL-15 human glioblastoma cell line. Macroscopic IKCa currents on GL-15 cells displayed a mean amplitude of 7.2±0.8 pA/pF at 0 mV, at day 1 after plating. The current was inhibited by clotrimazole (CTL, IC 50=257 nM), TRAM-34 (IC50=55 nM), and charybdotoxin (CTX, IC50=10.3 nM). RT-PCR analysis demonstrated the expression of mRNA encoding the IKCa channel in GL-15 cells. Unitary currents recorded using the inside-out configuration had a conductance of 25 pS, a KD for Ca2+ of 188 nM at -100 mV, and no voltage dependence. We tested whether the IKCa channel expression in GL-15 cells could be the result of an increased ERK activity. Inhibition of the ERK pathway with the MEK antagonist PD98059 (25 μM, for 5 days) virtually suppressed the IK Ca current in GL-15 cells. PD98059 treatment also increased the length of cellular processes and up-regulated the astrocytic differentiative marker GFAP. A significant reduction of the IKCa current amplitude was also observed with time in culture, with mean currents of 7.17±0.75 pA/pF at 1-2 days, and 3.11±1.35 pA/pF at 5-6 days after plating. This time-dependent down-regulation of the IKCa current was not accompanied by changes in the ERK activity, as assessed by immunoblot analysis. Semiquantitative RT-PCR analysis demonstrated a ∼35% reduction of the IKCa channel mRNA resulting from ERK inhibition and a ∼50% reduction with time in culture.
KW - Cell differentiation
KW - ERK
KW - GFAP
KW - Human glioblastoma GL-15 cell line
KW - Intermediate-conductance Ca-activated K channels
UR - http://www.scopus.com/inward/record.url?scp=33748990315&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33748990315&partnerID=8YFLogxK
M3 - Article
C2 - 16914889
AN - SCOPUS:33748990315
VL - 18
SP - 47
EP - 56
JO - Cellular Physiology and Biochemistry
JF - Cellular Physiology and Biochemistry
SN - 1015-8987
IS - 1-3
ER -