Stimulation of Na+-alanine cotransport activates a voltage-dependent conductance in single proximal tubule cells isolated from frog kidney.
1. The swelling induced by Na+-alanine cotransport in proximal tubule cells of the frog kidney is followed by regulatory volume decrease (RVD). This RVD is inhibited by gadolinium (Gd3+), an inhibitor of stretch-activated channels, but is independent of extracellular Ca2+. 2. In this study, the whole cell patch clamp technique was utilized to examine the effect of Na+-alanine cotransport on two previously identified volume- and Gd3+-sensitive conductances. One conductance is voltage dependent and anion selective (GVD) whilst the other is voltage independent and cation selective (GVI). 3. Addition of 5 mM L-alanine to the bathing solution increased the whole cell conductance and gave a positive (depolarizing) shift in the reversal potential (Vrev, equivalent to the membrane potential in current-clamped cells) consistent with activation of Na+-alanine cotransport. Vrev shifted from -36 +/- 4.9 to +12.9 +/- 4.2 mV (n = 15). 4. In the presence of alanine, the total whole cell conductance had several components including the cotransporter conductance and GVD and GVI. These conductances were separated using Gd3+, which inhibits both GVD and GVI, and the time dependency of GVD. Of these two volume-sensitive conductances, L-alanine elicited a specific increase in GVD, whereas GVI was unaffected. 5. The L-alanine-induced activation of GVD was significantly reduced when cells were incubated in a hypertonic bathing solution. 6. In summary, in single proximal tubule cells isolated from frog kidney, on stimulation of Na+-alanine cotransport GVD is activated, while GVI is unaffected. Taken with other evidence, this suggests that GVD is activated by cell swelling, consequent upon alanine entry, and may play a role as an anion efflux pathway during alanine-induced volume regulation.
Robson
L
L
Department of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, UK. l.robson@sheffield.ac.uk
Hunter
M
M
D000409:Alanine / Q000378:metabolism*; D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D004594:Electrophysiology; D005682:Gadolinium / Q000494:pharmacology; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials; D009997:Osmotic Pressure; D018408:Patch-Clamp Techniques; D011896:Rana temporaria; D012964:Sodium / Q000378:metabolism*; D027981:Symporters
D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D027981:Symporters; C052168:sodium-alanine cotransporter; D012964:Sodium; D005682:Gadolinium; D000409:Alanine