Cl- channels in basolateral TAL membranes: XIII. Heterogeneity between basolateral MTAL and CTAL Cl- channels.
BACKGROUND
Antidiuretic hormone (ADH) or adenosine 3', 5'-cyclic phosphate (cAMP) analogues augment net NaCl absorption in microperfused mouse medullary thick ascending limb (MTAL) segments but not in cortical thick ascending limb (CTAL) segments. This ADH-dependent MTAL effect is due to increased apical Na+/K+/2Cl- admittance and apical K+ recycling accompanied by a rise in calculated intracellular Cl- concentrations and by a threefold rise in basolateral Cl- conductance. rbClC-Ka, a 75.2 member of the ClC family of Cl- channels, mediates net Cl- absorption in the MTAL. The gating characteristics of rbClC-Ka channels from their intracellular surfaces are, to our knowledge, unique among Cl- channels. The channels are activated by small increases in intracellular Cl- (K1/2 = 10 mM Cl-). Adenosine triphosphate plus the catalytic subunit of protein kinase A (ATP + PKA) gate rbClC-Ka when cytosolic Cl- concentrations are 25 mM. Thus, in mouse MTAL segments, ADH-dependent rises in cytosolic Cl- are primarily responsible for basolateral Cl- conductance increases.


METHODS
These experiments compared the properties of Cl- channels fused into bilayers from basolaterally enriched vesicles from cultured mouse CTAL cells with rbClC-Ka channels.


RESULTS
The key findings were that anti-rbClC-Ka, antibody that recognizes and blocks rbClC-Ka, recognized and blocked basolateral Cl- channels in CTAL cells, that the extracellular faces of the CTAL channels were, like rbClC-Ka, substrate gated with a K1/2 of approximately 170 mM Cl-, and that, unlike rbClC-Ka channels, cytosolic faces of basolateral CTAL Cl- channels were not gated by either increasing cytosolic Cl- concentrations or cytosolic (ATP + PKA). This failure of activation of basolateral CTAL Cl- channels was confirmed using excised patch clamp studies. Finally, on Western blots, anti-rbClC-Ka recognized a 74 kDa band on basolateral CTAL vesicles.


CONCLUSIONS
Basolateral CTAL Cl- channels probably share a high degree of structural homology and possibly molecular mass with rbClC-Ka channels. However, significant differences between rbClC-Ka channels and CTAL Cl- channels account for the inability of increasing either cytosolic Cl- or (PKA + ATP) to raise Po in CTAL basolateral Cl- channels.
Winters
C J
CJ
Department of Internal Medicine, University of Arkansas College of Medicine, Little Rock, USA.
Reeves
W B
WB
Andreoli
T E
TE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002478:Cells, Cultured; D018118:Chloride Channels / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D002712:Chlorides / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004594:Electrophysiology; D007425:Intracellular Membranes / Q000378:metabolism*; D007672:Kidney Cortex; D007679:Kidney Medulla; D008138:Loop of Henle / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques