Thyroid Hormone Beta Receptor Mutation Causes Renal Dysfunction and Impairment of ClC-2 Chloride Channel Expression in Mouse Kidney

The voltage-dependent ClC-2 chloride channel has been implicated in a variety of physiological functions, including fluid transport across specific epithelia. ClC-2 is activated by hyperpolarization, weakly acidic external pH, intracellular Cl−, and cell swelling. To add more insight into the mechanisms involved in ClC-2 regulation, we searched for associated proteins that may influence ClC-2 activity. With the use of immunoprecipitation of ClC-2 from human embryonic kidney-293 cells stably expressing the channel, followed by electrophoretic separation of coimmunoprecipitated proteins and mass spectrometry identification, Hsp70 and Hsp90 were unmasked as possible ClC-2 interacting partners. Association of Hsp90 with ClC-2 was confirmed in mouse brain. Inhibition of Hsp90 by two specific inhibitors, geldanamycin or radicicol, did not affect total amounts of ClC-2 but did reduce plasma membrane channel abundance. Functional experiments using the whole cell configuration of the patch-clamp technique showed that inhibition of Hsp90 reduced ClC-2 current amplitude and impaired the intracellular Cl− concentration [Cl−]-dependent rightward shift of the fractional conductance. Geldanamycin and radicicol increased both the slow and fast activation time constants in a chloride-dependent manner. Heat shock treatment had the opposite effect. These results indicate that association of Hsp90 with ClC-2 results in greater channel activity due to increased cell surface channel expression, facilitation of channel opening, and enhanced channel sensitivity to intracellular [Cl−]. This association may have important pathophysiological consequences, enabling increased ClC-2 activity in response to cellular stresses such as elevated temperature, ischemia, or oxidative reagents.

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Perinatal regulation of the ClC-2 chloride channel in lung is mediated by Sp1 and Sp3

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.276.4.l614 ◽

1999 ◽

Vol 276 (4) ◽

pp. L614-L624 ◽

Cited By ~ 10

Author(s):

Shijian Chu ◽

Carol J. Blaisdell ◽

Min-Zhi M. Liu ◽

Pamela L. Zeitlin

Keyword(s):

Chloride Channel ◽

Fetal Lung ◽

Airway Disease ◽

Perinatal Period ◽

Lung Epithelial Cells ◽

Normal Lung ◽

Mobility Shift ◽

Channel Expression ◽

Gc Box ◽

Electrophoretic Mobility Shift Assays

Mechanisms responsible for regulation of pulmonary epithelial chloride-channel expression in the perinatal period are under investigation to better understand normal lung development and airway disease pathogenesis. The ClC-2 epithelial chloride channel is regulated by changes in pH and volume and is most abundant in lung during fetal development. In this study, we identify and sequence the ClC-2 promoter, which is GC rich and lacks a TATA box. By construction of a series of promoter-luciferase constructs, a 67-bp GC box-containing sequence in the promoter is shown to be critical to ClC-2 expression in primary and immortalized fetal lung epithelial cells. Electrophoretic mobility shift assays and antibody supershifts demonstrate that the Sp1 and Sp3 transcription factors are expressed in fetal lung nuclei and interact with the GC box sequences in the promoter. Immunoblotting techniques demonstrate that Sp1 and Sp3 are perinatally downregulated in the lung with the same temporal sequence as ClC-2 downregulation. This work suggests that Sp1 and Sp3 activate ClC-2 gene transcription and that reduction in Sp1 and Sp3 at birth explains perinatal downregulation of ClC-2 in the lung.

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Inhibition of CLC-2 chloride channel expression interrupts expansion of fetal lung cysts

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00113.2003 ◽

2004 ◽

Vol 286 (2) ◽

pp. L420-L426 ◽

Cited By ~ 12

Author(s):

Carol J. Blaisdell ◽

Marcelo M. Morales ◽

Ana Carolina Oliveira Andrade ◽

Penelope Bamford ◽

Michael Wasicko ◽

...

Keyword(s):

Chloride Channel ◽

Fluid Transport ◽

Fetal Lung ◽

Chloride Secretion ◽

Normal Lung ◽

Apical Surface ◽

Lung Cysts ◽

Lung Fluid ◽

Lung Morphogenesis ◽

Channel Expression

Normal lung morphogenesis is dependent on chloride-driven fluid transport. The molecular identity of essential fetal lung chloride channel(s) has not been elucidated. CLC-2 is a chloride channel, which is expressed on the apical surface of the developing respiratory epithelium. CLC-2-like pH-dependent chloride secretion exists in fetal airway cells. We used a 14-day fetal rat lung submersion culture model to examine the role of CLC-2 in lung development. In this model, the excised fetal lung continues to grow, secrete fluid, and become progressively cystic in morphology ( 26 ). We inhibited CLC-2 expression in these explants, using antisense oligonucleotides, and found that lung cyst morphology was disrupted. In addition, transepithelial voltage ( Vt) of lung explants transfected with antisense CLC-2 was inhibited with Vt = -1.5 ± 0.2 mV (means + SE) compared with -3.7 ± 0.3 mV (means + SE) for mock-transfected controls and -3.3 ± 0.3 mV (means + SE) for nonsense oligodeoxynucleotide-transfected controls. This suggests that CLC-2 is important for fetal lung fluid production and that it may play a role in normal lung morphogenesis.

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Hypothyroidism does not affect the dihydropyridine sensitivity of precontracted murine uterus

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y03-090 ◽

2003 ◽

Vol 81 (9) ◽

pp. 890-893 ◽

Cited By ~ 3

Author(s):

Jörg W Wegener ◽

Matthias Lee ◽

Franz Hofmann

Keyword(s):

Smooth Muscle ◽

Cell Differentiation ◽

Thyroid Hormone ◽

Thyroid Hormones ◽

Rat Uterus ◽

Hormone Levels ◽

Uterine Muscle ◽

Channel Expression ◽

Thyroid Hormone Levels ◽

Pregnant State

Thyroid hormones are known to influence various processes of cell differentiation. Recently, it was reported that hypothyroidism reduces the sensitivity to Ca2+-channel antagonists in the rat uterus. We examined the sensitivity to dihydropyridines of the uterus from mice that had reduced thyroid hormone levels. Isradipine relaxed with the same potency precontracted uterine muscle strips from control and hypothyroid mice, independently from a pseudo-pregnant state. These results demonstrate that hypothyroidism does not change dihydropyridine sensitivity (i.e., the pattern of Ca2+-channel expression) in the murine uterus.Key words: uterus, smooth muscle, Ca2+ channel, isradipine.

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