K+ channel cAMP activated in guinea pig gallbladder epithelial cells.
In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patch-clamp techniques whether K(+) channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskolin or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (P(o)) values for Ca(2+)-activated K(+) channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in P(o) of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (10(-5) M) was able to reduce P(o) when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the alpha subunit of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments confirm that these cells express the BK alpha subunit, which is located on the apical membrane. Other K(+) channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA + ATP + cAMP (inside-out). These channels were insensitive to TEA(+) and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca(2+)-dependent, loperamide-sensitive K(+) channels present on the apical membrane.
Meyer
Giuliano
G
Dipartimento di Fisiologia e Biochimica Generali, Sezione di Fisiologia Generale, Università degli Studi di Milano, Via Celoria 26, Milan I-20133, Italy. Giuliano.Meyer@unimi.it
Bazzini
Claudia
C
Bottà
Guido
G
Garavaglia
Maria Lisa
ML
Simona
Rodighiero
R
Manfredi
Rosangela
R
Sironi
Chiara
C
De Biasi
Silvia
S
Paulmichl
Markus
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002469:Cell Separation; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D004594:Electrophysiology / Q000379:methods; D004847:Epithelial Cells / Q000378:metabolism*; D005704:Gallbladder / Q000378:metabolism*; D006168:Guinea Pigs; D007150:Immunohistochemistry; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques / Q000379:methods; D024681:Potassium Channels, Calcium-Activated / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D000242:Cyclic AMP