Ion channels in endosomal membranes from rabbit kidney cortex were studied after reconstitution into planar lipid bilayers. The most frequently observed ion channel was anion selective (P(Cl)/P(K) = 13) and had a single-channel conductance of 116 pS when the cis and trans solutions contained 410 and 150 mM KCl, respectively, and a conductance of 90 pS in symmetrical 150 mM KCl solutions. The anion selectivity sequence of the channel was NO3/- > F- > Br- > Cl- >> I-. The activity of the channel was voltage dependent such that hyperpolarization of the cis, or cytoplasmic, surface of the channel increased the open probability (P(o)). The activity of the channel was also highly dependent on the calcium activity of the cis but not the trans solution. Channels were fully active (P(o) > 0.7) at Ca2+ concentration > 1 μM, but channel activity was completely absent (P(o) < 0.001) at Ca2+ concentration < 250 nM. The effects of calcium on P(o) were not voltage dependent. The Cl--channel blocker 2-[(2-cyclopentyl-6,7-dichloro-2,3- dihydro-2-methyl-1-oxo-1H-inden-5-yl)oxy]-acetic acid (IAA-94/95) produced a concentration-dependent reversible flickering block of the endosomal channel with a K(i) of 15 μM. 4,4'-Dinitrostilbene-2,2'-disulfonic acid, a disulfonic stilbene, also produced a flickering block of the channel with a K(i) of ~5 μM. Because endosomal Cl- channels are believed to facilitate endosomal acidification, we tested the effects of IAA-94/95 and deletion of Ca2+ on the rate of acidification of intact endosomes. Because neither maneuver affected acidification, we conclude that the 116-pS channel does not participate in endosomal acidification. This channel may be involved in other endosomal processes, e.g., cell volume regulation and control of membrane trafficking.
- 4,4'-dinitrostilbene-2,2'disulfonic acid
ASJC Scopus subject areas
- Cell Biology