Abstract
The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) is targeted to the basolateral membrane in polarized epithelia where it plays an essential role in promoting trans-epithelial ion transport. KCa3.1 is found in many tissues in the body and plays an important role in many physiological and pathological processes (e.g., regulation of salt and fluid transport in the gastrointestinal tract, atherosclerosis, sickle cell disease and asthma).
Functional KCa3.1 must be targeted to the basolateral membrane, a process that is dependent upon proper cytoskeletal function. The cytoskeleton is comprised of actin and microtubule filaments. Actin filaments are comprised of polymerised G-actin monomers bound to form filamentous F-actin strands. Microtubules are long filamentous structures comprised of tubulin subunits, made from α-tubulin and β-tubulin monomers.
This study examines the role of microfilaments and microtubules in the trafficking of KCa3.1 to the basolateral membrane of polarised epithelial cells. To address this, Fischer Rat Thyroid cells grown on filter inserts to form a confluent epithelium were stably transfected with the Biotin Ligase Acceptor Peptide (BLAP)-KCa3.1 construct. This construct allowed for the selective labeling of basolaterally expressed KCa3.1 using streptavidin. Selective labeling of membrane bound KCa3.1 allowed for the measurement of changes in KCa3.1 expression, in response to drugs that disrupt cytoskeletal elements, to reflect changes in KCa3.1 located on the basolateral membrane. This measure allowed for a direct correlation to be drawn between targeted disruption of specific cytoskeletal elements, e.g. microtubules and microfilaments, and expression of basolaterally-located KCa3.1.
PCR was used to determine the mRNA expression levels of KCa3.1 in stably transfected cell lines and SDS-PAGE techniques were employed to investigate protein expression levels of KCa3.1. Western blotting was used to explore the effects of Cytochalasin D (Cyto D), Latrunculin A (Lat A), and Myosin Light Chain Inhibitor-7 (ML-7) which inhibit the function of actin (Cyto D, Lat A) and myosin light chain kinase (ML-7) respectively.
Toxicity tests were performed to determine cell survival under a range concentrations of 0-20 μM (0, 3, 5 hr) for all three drugs with cell survival reduced with 20 μM at t = 5 hr for Cyto D and Lat A.
Cyto D was administered over intervals of 0, 3 and 5 hr at 10 μM resulting in a decreased relative expression of KCa3.1 (compared to control) of 0.6±0.14 at t = 3 and further decrease in the expression of the channel at t = 5 hr with a relative expression of 0.12±0.035 (n = 5, p < 0.05). Lat A was also administered over intervals of 0, 3 and 5 hr at 10 μM causing a relative reduction in the expression of KCa3.1 at the basolateral membrane compared to the control. At t = 3 hr the expression of KCa3.1 was reduced to 0.7±0.065 and decreased to 0.3±0.049 at t = 5 hr (n = 4, p < 0.001). Finally, cells treated with microtubule inhibitor ML-7 showed a relative reduction in KCa3.1 expression of 0.55±0.12 at t = 3 hr, the expression was further decreased to 0.33±0.11 at t = 5 hr compared to the control.
These data confirm that microtubules and microfilaments of the cytoskeleton are crucial in trafficking KCa3.1 to the basolateral membrane of polarised epithelial cells.