cAMP-activated chloride channel in the basolateral membrane of the thick ascending limb of the mouse kidney

Abstract

The properties of an anion-selective channel observed in basolateral membranes of microdissected, collagenase-treated, cortical thick ascending limbs of Henle's loop from mouse kidney were investigated using patch-clamp single-channel recording techniques. In basal conditions, single Cl⁻ currents were detected in 8% of cell-attached and excised, inside-out, membrane patches whereas they were observed in 24% of cell-attached and 67% of inside-out membrane patches when tubular fragments were preincubated with Forskolin (10⁻⁵ m) or 8-bromo-cAMP (10⁻⁴ m) and isobutylmethylxanthine (10⁻⁵ m). The channel exhibited a linear current-voltage relationship with conductances of about 40 pS in both cell-attached and cell-free membrane configurations. AP _Na ⁺ P _Cl ⁻ratio of 0.05 was estimated in the presence of a 142/42mm NaCl concentration gradient applied to inside-out membrane patches. Anionic selectivity of the channel followed the sequence Cl⁻>Br⁻>No ₃ ⁻ ≫F⁻; gluconate was not a permeant species. The open-state probability of the channel increased with membrane depolarization in cell-attached, i.e.,in situ membrane patches. In excised, inside-out, membrane patches, the channel was predominantly open with the open-state probability close to 0.8 over the whole range of potentials tested (−60 to +60 mV). The channel activity was not a function of internal calcium concentration between 10⁻⁹ and 10⁻³ m. We suggest that this Cl⁻ channel, whose properties are distinct from those in other epithelia, could account for the well-documented conductance which mediates Cl⁻ exit in the basolateral step of NaCl absorption in thick ascending limb of Henle's loop.