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 G ，Bazzini C ，Bottà G ，Garavaglia ML ，Simona R ，Manfredi R ，Sironi C ，De Biasi S ，Paulmichl M ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

An anion channel in guinea pig gallbladder epithelial cells is highly permeable to HCO(-)(3).

In guinea pig gallbladder epithelium, a secretion of fluid, secondary to an electrogenic secretion of Cl(-) and HCO(-)(3), is elicited in the presence of a high intracellular concentration of adenosine 3'-5'-cyclic monophosphate (cAMP). The aim of this study was to analyze the effects of secretagogues on the activity of anionic channels in isolated epithelial cells using the patch-clamp technique and measuring the electrical potential difference of the cellular membrane (pd(cm)). In cell-attached configuration, with the microelectrode filled with a solution of N-methylglucamine-Cl, or in inside-out configuration (symmetrical solution), it was possible to demonstrate the presence of an 18-pS Cl(-) channel with linear current/voltage (I/V) relationship and voltage independence; this channel is not activated by cAMP (cell-attached configuration). In inside-out configuration (symmetrical solution), another anionic channel with a conductance of 2.8 pS, voltage independence, and a linear I/V relationship was also identified. This channel was stimulated by cAMP (cell-attached configuration) and by PKA + ATP + cAMP (inside-out configuration). The channel was inhibited by NPPB (10(-5) M), but not by other anionic inhibitors. Measurements of the pd(cm) value suggested that in isolated cells, as in whole tissue, cAMP activates conductance for both Cl(-) and HCO(-)(3). The selectivity of the channel was gluconate < SO(2-)(4) < Cl(-) < Br(-) < I(-) < HCO(-)(3) < SCN(-) and the P(HCO(3))/P(Cl) was 2.6. Some features of the channel resemble those of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and RT-PCR performed on mRNA from isolated epithelial cells detected the presence of a CFTR homologue mRNA. The results obtained indicate that this channel is responsible for the HCO(-)(3) conductance activated by cAMP.

Meyer G ，Garavaglia ML ，Bazzini C ，Bottà G ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Molecular and functional identification of cyclic AMP-sensitive BKCa potassium channels (ZERO variant) and L-type voltage-dependent calcium channels in single rat juxtaglomerular cells.

Friis UG ，Jørgensen F ，Andreasen D ，Jensen BL ，Skøtt O ... -  《-》

Expression of Ca2+-activated BK channel mRNA and its splice variants in the rat cochlea.

Voltage-activated K(+) channels are important for shaping the receptor potentials of cochlear hair cells. In particular, the functional maturation of inner hair cells in mice around the onset of hearing coincides with the expression of a large, fast K(+) conductance, probably mediated by Ca(2+)-activated K(+) (BK) channels. In hearing organs of lower vertebrates, frequency tuning depends on BK-type K(+) channels with different kinetics. Kinetics are varied by alternative splicing of the channels' alpha subunits and combination with modulating beta subunits. It is unclear whether similar mechanisms "fine tune" mammalian hair cells. We used various polymerase chain reaction (PCR) approaches to screen rat cochleae for splice variants of BK-type alpha subunits. We isolated mainly minimal variants and only occasionally splice variants with additional inserts. We conclude that alpha subunits with different kinetics are not substantially used in the rat cochlea. However, we isolated six variants differing in their extreme C-terminal sequences, which may be involved in the targeting of the channel protein. By using reverse transcriptase-PCR, we demonstrated also the expression of transcripts for several beta subunits. In situ hybridization experiments revealed strict coexpression of alpha with beta1 transcripts. In inner hair cells, strong labeling emerged shortly before the onset of hearing. Labeling of outer hair cells appeared later and generally weaker. Thus, our molecular data confirm electrophysiological results that suggested that BK channels underlie the large K(+) conductance in inner hair cells of mammals. Extensive splicing of BK channel transcripts, however, does not seem to be used in mammalian hair cells as is done in lower vertebrates.

Langer P ，Gründer S ，Rüsch A 《JOURNAL OF COMPARATIVE NEUROLOGY》

Human podocytes possess a stretch-sensitive, Ca2+-activated K+ channel: potential implications for the control of glomerular filtration.

Morton MJ ，Hutchinson K ，Mathieson PW ，Witherden IR ，Saleem MA ，Hunter M ... -  《JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY》