TY - JOUR
T1 - Cl- channels in basolateral TAL membranes
T2 - XIII. Heterogeneity between basolateral MTAL and CTAL Cl- channels
AU - Winters, Christopher J.
AU - Reeves, W. Brian
AU - Andreoli, Thomas E.
N1 - Funding Information:
This work was supported by a National Institutes of Health Grant (5 R01 DK25540) and a Veterans Administration Merit Review Grant to T.E. Andreoli. We are very grateful to Dr. Ludwika Zimniak for valuable dialogue. The technical assistance provided by Anna Grace Stewart and the secretarial assistance provided by Clementine Whitman are greatly appreciated. W.B. Reeves is an established investigator of the American Heart Association (95-1450).
PY - 1999
Y1 - 1999
N2 - 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- (K( 1/2 ) = 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, an 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 K( 1/2 ) 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 P(o) in CTAL basolateral Cl- channels.
AB - 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- (K( 1/2 ) = 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, an 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 K( 1/2 ) 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 P(o) in CTAL basolateral Cl- channels.
KW - Chloride channels
KW - Potassium recycling
KW - Sodium chloride
KW - Thick ascending limb
KW - rbClC-Ka
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U2 - 10.1046/j.1523-1755.1999.00270.x
DO - 10.1046/j.1523-1755.1999.00270.x
M3 - Article
C2 - 9987083
AN - SCOPUS:0032933189
SN - 0085-2538
VL - 55
SP - 593
EP - 601
JO - Kidney international
JF - Kidney international
IS - 2
ER -