Expression and modulation of the intermediate- conductance Ca2+-activated K+ channel in glioblastoma GL-15 cells.
We report here the expression and properties of the intermediate-conductance Ca(2+)-activated K(+) (IK(Ca)) channel in the GL-15 human glioblastoma cell line. Macroscopic IK(Ca) 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 (IC(50)=55 nM), and charybdotoxin (CTX, IC(50)=10.3 nM). RT-PCR analysis demonstrated the expression of mRNA encoding the IK(Ca) channel in GL-15 cells. Unitary currents recorded using the inside-out configuration had a conductance of 25 pS, a K(D) for Ca(2+) 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 muM, 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 downregulation of the IK(Ca) 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 IK(Ca) channel mRNA resulting from ERK inhibition and a approximately 50% reduction with time in culture.
Fioretti
Bernard
B
Dipartimento di Biologia Cellulare e Ambientale, Universita' di Perugia, CEMIN, Centro di Eccellenza "Materiali Innovativi Nanostrutturati", Universita' di Perugia, Italy. fabiolab@unipg.it
Castigli
Emilia
E
Micheli
Maria R
MR
Bova
Rodolfo
R
Sciaccaluga
Miriam
M
Harper
Alexander
A
Franciolini
Fabio
F
Catacuzzeno
Luigi
L
D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D005419:Flavonoids / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D005909:Glioblastoma / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D006801:Humans; D015151:Immunoblotting; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques / Q000379:methods; D026902:Potassium Channel Blockers / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013757:Tetraethylammonium Compounds / Q000494:pharmacology; D013997:Time Factors
D005419:Flavonoids; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D026902:Potassium Channel Blockers; D012333:RNA, Messenger; D013757:Tetraethylammonium Compounds; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one