Ca2+ regulated K+ and non-selective cation channels in the basolateral membrane of rat colonic crypt base cells

Abstract

We have previously shown that a new type of K+ channel, present in the basolateral membrane of the colonic crypt base (blm), is necessary for cAMP-activated Cl- secretion. Under basal conditions, and when stimulated by carbachol (CCH) alone, this channel is absent. In the present patch clamp-study we examined the ion channels present in the blm under cell-attached and in cell-excised conditions. In cell-attached recordings with NaCl-type solution in the pipette we measured activity of a K+ channel of 16 +/- 0.3 pS (n = 168). The activity of this channel was sharply increased by CCH (0. 1 mmol/l, n = 26). Reduction of extracellular Ca2+ to 0.1 mmol/l (n = 34) led to a reversible reduction of activity of this small channel (SKCa). It was also inactivated by forskolin (5 micromol/l, n = 38), whilst the K+ channel noise caused by the very small K+ channel increased. Activity of non-selective cation channels (NScat) was rarely observed immediately prior to the loss of attached basolateral patches and routinely in excised patches. The NScat, with a mean conductance of 49 +/- 1.0 pS (n = 96), was Ca2+ activated and required >10 micromol/l Ca2+ (cytosolic side = cs). It was reversibly inhibited by ATP (<1 mmol/l, n = 13) and by 3',5-dichloro-diphenylamine-2-carboxylate (10-100 micromol/l, n = 5). SKCa was also Ca2+ dependent in excised inside-out basolateral patches. Its activity stayed almost unaltered down to 1 micromol/l (cs) and then fell sharply to almost zero at 0.1 micromol/l Ca2+ (cs, n = 12). SKCa was inhibited by Ba2+ (n = 31) and was charybdotoxin sensitive (1 nmol/l) in outside-out basolateral patches (n = 3). Measurements of the Ca2+ activity ([Ca2+]i) in these cells using fura-2 indicated that forskolin and depolarization, induced by an increase in bath K+ concentration to 30 mmol/l, reduced [Ca2+]i markedly (n = 8-10). Hyperpolarization had the opposite effect. The present data indicate that the blm of these cells contains a small-conductance Ca2+-sensitive K+ channel. This channel is activated promptly by very small increments in [Ca2+]i and is inactivated by a fall in [Ca2+]i induced by forskolin.

Tissue specific ionomotive enzyme activity and K+ reabsorption reveal the rectum as an important ionoregulatory organ in larvalChironomous ripariusexposed to varying salinity

“…The dose of ChTX was selected such that it equalled the highest concentration used in other insect and mammalian tissue studies (Miller et al, 1985;MacKinnon et al, 1988;Grinstein and Smith, 1990;Bleich et al, 1996). Ouabain and bafilomycin were dissolved in DMSO (Sigma-Aldrich) and, prior to use, diluted to a desired concentration in saline (composition defined above in Immunohistochemical localization of NKA and VA).…”

“…ChTX is a small basic protein purified from the venom of the scorpion Leiurus quinquestriatus (Smith et al, 1986). It has been shown to block both large-and small-conductance Ca 2+ -activated K + channels as well as Ca 2+ -insensitive, voltage-dependent K + channels (Miller et al, 1985;Hermann and Erxleben, 1987;MacKinnon et al, 1988;Grinstein and Smith, 1990;Bleich et al, 1996). Interestingly, expression of a gene encoding the Ca + -activated K + channel has been localized in the ion-transporting midgut epithelial cells of Drosophila (Brenner and Atkinson, 1997).…”

Distal Colonic K+ Secretion Occurs via BK Channels

“…The SK4 channel is well documented to be localized in the basolateral membrane of rodent colonic crypts (18,20 …”

P2Y6 receptor mediates colonic NaCl secretion via differential activation of cAMP-mediated transport

“…shown to drive secretion (13)(14)(15). A typical secretory agonist such as PGE 2 elevates cAMP, which activates luminal CFTR and basolateral KCNQ1/KCNE3 channels (11).…”