Voltage and Ca2+-Activated K+ channel in cultured epithelial cells (MDCK)

Abstract

Patch-clamp techniques were used to study a K channel in the cell membrane of MDCK cells. This cell line derives from the kidney of a normal dog, presumably from the distal nephron, a region involved in potassium secretion. The cells were cultured in confluent monolayers and approached from the apical side. The K channel we describe is Ca2+ and voltage activated, has a conductance of 221 +/- 7 pS, and can be inhibited by 10 mM tetraethylammonium and by 1 mM quinidine, but not by 4-aminopyridine, nor by 1 mM Ba2+ added to the outer side. Using the whole-cell configuration, we find that most of the cationic conductance of the membrane is constituted by a K-specific one (maximum K conductance 32.1 +/- 3.9 nS vs. a leak conductance of 1.01 +/- 0.17 nS). Comparisons of the maximum K conductance with that of a single K channel indicates that an MDCK cell has an average of 145 such channels. The membrane capacity is 24.5 +/- 1.4 pF.

Apical sorting of a voltage- and Ca 2+ -activated K + channel α-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation

“…Apical sorting information in the K V,Ca ␣-subunit explains its electrophysiological detection in the apical domain of epithelial cells from kidney (27)(28)(29), male reproductive tract (30), oviduct (31), and vestibulum (32), but leaves uncertain the mechanism of its basolateral distribution reported in enterocytes (33) and epithelial cells from acinar glands (34). Tissue-specific expression of hSlo splice variants might confer variability in the functional expression or decoding of targeting signals, as reported for K v channels (18).…”

“…1B). This strategy to study hSlo channel sorting was used because the number of hSlo channels in MDCK cells has been estimated as 145 per cell (27). This amount is under the limit of detection by techniques currently used to study polarity at the protein level.…”

“…The ␤-subunits are N-glycosylated and, with the exception of a ␤3 isoform, endow the channel with altered kinetics, calcium͞voltage, and toxin sensitivities (23)(24)(25). In a diversity of epithelial cells, from kidney (27)(28)(29), male reproductive tract (30), oviduct (31), and vestibulum (32), K V,Ca channels have been electrophysiologically detected at the apical domain. However, they have been also reported in the basolateral domain of enterocytes (33) and epithelial cells from acinar glands (34).…”

“…In Madin-Darby canine kidney (MDCK) cells, which presumably are derived from the transporting kidney epithelial cells of the distal tubule and early portion of the collecting duct, the K V,Ca channels have been detected only apically by electrophysiology (27,28). We have overexpressed recombinant K V,Ca channel ␣-subunit (human Slowpoke channel, hSlo) bearing an N-terminal c-myc epitope in permanently transfected MDCK cells to assess its regionalization and molecular sorting mechanisms at the protein level.…”

The Cytoplasmic Tail of Large Conductance, Voltage- and Ca2+-activated K+ (MaxiK) Channel Is Necessary for Its Cell Surface Expression

“…Structures unique to the MaxiK channel ␣-subunit include an additional N-terminal transmembrane domain, S0, and a large C terminus. There are four hydrophobic segments (S7-S10) in the C-terminal region of the channel protein (21,23). These segments were previously thought to be additional transmembrane domains, but now it is agreed that they are located inside the cell (21).…”