The role of the microtubule cytoskeleton and Myosin-Vc in the anterograde trafficking of KCa3.1 to the basolateral membrane of polarised epithelial cells.
The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) is targeted to the basolateral membrane in polarised epithelia where it plays an essential role in promoting trans-epithelial ion transport. KCa3.1 is expressed throughout the body and plays a critical role in physiological processes such as the regulation of salt and fluid transport and the maintenance of membrane potential and the electrochemical gradient required for typical cell function. Considering the wide and varied physiological roles of KCa3.1, it is unsurprising that in the last 20 years KCa3.1 has emerged as a key therapeutic target for many diseases ranging from neuropathy to asthma to xerocytosis. In this study I investigated two key molecular components involved in the anterograde trafficking of KCa3.1 to the basolateral membrane of polarised epithelial cells – the microtubule cytoskeleton, and actin motor protein Myosin-Vc. To address this, Fischer Rat Thyroid cells stably transfected with a biotin-ligase-acceptor peptide (BLAP)-KCa3.1 sequence. FRT-KCa3.1-BLAP epithelia are cultured on Transwell and Snapwell inserts to form a polarised epithelial monolayer. KCa3.1 expressed by FRT-KCa3.1-BLAP epithelia was selectively labelled at the basolateral membrane with streptavidin. Immunoblot and Ussing chamber techniques allowed for the study of the membrane expression of the channel and consequent measured changes in response to microtubule and Myosin inhibiting drugs and the knock down of Myosin-Vc. The microtubule cytoskeleton is comprised of polarised, dynamic, and hollow tube shaped structures formed from tight helices of polymerised dimers of alpha and beta tubulin. To determine the role of microtubules in the trafficking of KCa3.1, microtubule function was pharmacologically inhibited with colchicine and the effect of this on KCa3.1 expression was investigated via immunoblot and Ussing chamber techniques. FRT-KCa3.1-BLAP epithelia treated with colchicine (10 μM) demonstrated KCa3.1 expression was reduced by 63±7% (**p≤0.01, n=5), and KCa3.1 K+ current was reduced by 54±19% (*p≤0.05, n=5) relative to untreated FRT-KCa3.1-BLAP epithelia. Previous data collected in the Hamilton lab has determined that microfilaments are required for the anterograde trafficking of KCa3.1. Myosin-V is a microfilament motor protein responsible for the anterograde trafficking of organelles and vesicles. Of particular interest to this study is Myosin-Vc which is highly expressed in epithelial tissues. Myosin-Vc was selected as a target motor protein for the trafficking of KCa3.1 after a series of Immunoblot and Ussing chamber experiments with broad spectrum pharmacological Myosin inhibitors. ML9 inhibits Myosin light chain kinase and inhibited the activity of Myosin light chain kinase regulated Myosins. FRT-KCa3.1-BLAP epithelia treated with ML9 (10 μM) demonstrated a reduced expression of basolateral KCa3.1 of 83±2% (**p≤0.01, n=5) with a KCa3.1 K+ current reduced by 54±2% (**P≤0.01, n=7) in cells treated with ML9 for 5 hr relative to untreated FRT-KCa3.1-BLAP epithelia. FRT-KCa3.1-BLAP epithelia were also treated with muscle and non-muscle Myosin-II and Myosin-V inhibitor BDM (10 mM) which reduced basolateral expression and activity of KCa3.1 by 58±5% (*p≤0.05, n=5) and 51±7% (**p≤0.01, n=8) respectively, relative to untreated FRT-KCa3.1-BLAP epithelia. Finally, Myosin-Vc was knocked down using Myosin-Vc specific siRNA decreasing KCa3.1 membrane expression by 78±9% (n=3, *p≤0.05) and KCa3.1 K+ conduction by 0.95±0.14 μA (n=3, *p≤0.05) relative to untransfected FRT-KCa3.1-BLAP epithelia. Based on immunoblot and Ussing chamber data, current findings suggest that the anterograde trafficking of KCa3.1 in a polarised epithelium is dependent on both the microtubule cytoskeleton and Myosin-Vc.
Advisor: Hamilton, Kirk L
Degree Name: Master of Science
Degree Discipline: Department of Physiology
Publisher: University of Otago
Keywords: Myosin-V; Myosin-Vc; Myosin 5; Myosin 2; Myosin-II; KCa3.1; colchicine; IK1; SK4; KCNN4; basolateral membrane; basolateral; epithelial; trafficking; microtubules; microtubule cytoskeleton; cytoskeleton; actin
Research Type: Thesis