Publication Date

2013

Document Type

Thesis

Committee Members

Norma Adragna (Advisor), Mauricio Di Fulvio (Committee Member), Kristopher Kahle (Committee Member), Peter Lauf (Committee Member)

Degree Name

Master of Science (MS)

Abstract

K-Cl cotransport (KCC) mediated by four protein isoforms, KCC1 to KCC4, plays a significant role in cell volume regulation, and in K and Cl homeostasis. In this study, we demonstrate the importance of KCC3 and its two threonine (T) phosphorylation sites T991 and T1048 in cellular K homeostasis in isogenic human embryonic kidney (HEK-293) cells transfected with KCC3 wild type (WT) or constitutively active double mutant KCC3 (AA) expressed under tetracycline or doxycycline control. In both WT and AA cells, under baseline conditions, the Na-K-2Cl cotransport (NKCC) (55 %- 60 %) was the major contributor of Rb influx followed by the Na/K pump (NKP) (35 % - 40 %) and KCC (10 % - 15 %) even though the absolute values of Rb influx were higher in AA cells. Doxycycline induction had no effect on either NKCC or KCC in WT cells, whereas NKCC was completely inhibited and KCC was increased by 25 fold in AA cells. In AA cells, intracellular K (Ki) decreased by 90 % upon doxycycline induction in Cl medium and 50 % of this loss was attenuated in Cl-free (sulfamate replacement) medium. Doxycycline-induced Cl-dependent Ki loss was sensitive to furosemide (2 mM) but insensitive to tetra ethyl ammonium (TEA) (2 mM) and to changes in external K and Na, whereas doxycycline-induced Cl-independent Ki loss was sensitive to 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB) (50 microM) but insensitive to TEA (2 mM), furosemide (2 mM) and extracellular K and Na.

Page Count

53

Department or Program

Department of Pharmacology and Toxicology

Year Degree Awarded

2013

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