Evidence of Decreased Activity in Intermediate-Conductance Calcium-Activated Potassium Channels During Retinoic Acid-Induced Differentiation in Motor Neuron-Like NSC-34 Cells.

BACKGROUND/AIMS: Intermediate-conductance Ca2+-activated K+ (IKCa; KCa3.1 or KCNN4) channels affect the behaviors of central neurons including motor neurons. The mechanism through which neuronal differentiation is related to the activity of these channels remains largely unclear. METHODS: By using various molecular biology tools and electrophysiological measurements, we investigated possible changes in the activity of IKCa channels in a retinoic acid (RA)-induced differentiation process in motor neuron-like NSC-34 cells. RESULTS: The protein and messenger RNA expression of KCa3.1 substantially diminished as NSC-34 cells were differentiated with low serum (1%) and 1 µM RA. In whole-cell current recordings, the density of delayed-rectifier K+ currents obtained from differentiated cells was elevated. However, the density of a ramp pulse-elicited K+ current that was sensitive to blockage by 1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole (TRAM-34)-an inhibitor of IKCa channels-was significantly higher in undifferentiated NSC-34 cells than in differentiated cells. In undifferentiated cells, the activity of IKCa channels was readily detected and the probability of channel openings was resistant to stimulation by diazoxide or suppression by verruculogen. Furthermore, this probability was increased by 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one or 9-phenanthrol and reduced by TRAM-34. The channel-opening probability decreased in RA-induced differentiated cells, whereas the single-channel conductance of IKCa channels did not differ between undifferentiated and differentiated cells. Moreover, the slow component of the mean closed time in these channels was significantly shorter in undifferentiated cells than in differentiated cells; however, the mean open time in the channel remained unchanged as cells were differentiated. CONCLUSION: RA-induced differentiation in neurons could exert a suppressive effect on the activity of IKCa channels.

Chronic palmitic acid-induced lipotoxicity correlates with defective trafficking of ATP sensitive potassium channels in pancreatic ß cells.

Lipotoxicity is associated with a high level of fatty acid accumulation in pancreatic ß-cells. An overload of free fatty acids contributes to pancreatic ß-cell apoptosis and dysfunction. Insulin secretion involves sequential ionic events upon glucose stimulation. ATP sensitive potassium (KATP) channels serve as glucose sensors and effectively initiate glucose-stimulated insulin secretion. This study investigated the effects of lipotoxicity on the trafficking of KATP channels in pancreatic ß cells using chronic palmitic acid -injected mice and treated insulinoma cells. The chronic palmitic acid -injected mice displayed type II diabetic characteristics. The pancreatic sections of these mice exhibited a decrease in the expression of KATP channels. We then tested the time and dose effects of palmitic acid on the cell viability of INS-1 cells. We observed a significant decrease in the surface expression of KATP channels after 72 h of treatment with 0.4 mM palmitic acid. In addition, this treatment induced pancreatic ß-cell apoptosis by increasing cleaved caspase 3 protein level. Our results demonstrated cotreatment with glibenclamide, the sulfonylurea compounds for type II diabetes mellitus, in palmitic acid -treated cells reduces cell death and recovers the glucose stimulated insulin secretion through increasing the surface expression of KATP channels. Importantly, glibenclamide also improved glucose tolerance, triglyceride concentration, and insulin sensitivity in the palmitic acid-injected mice. In conclusion, an increase in the surface expression of KATP channels restores insulin secretion, reduces pancreatic ß-cell's apoptosis, highlighting correct trafficking of KATP channels is important in survival of ß-cells during lipotoxicity.

Monitoring Changes in the Abundance of Endogenously Expressed ATP-Sensitive Potassium (KATP) Channels in the Plasma Membrane Using Surface Biotinylation.

The conductance of KATP channel activity can be regulated by gating and/or surface expression. Gating analysis is usually performed by electrophysiological recording. Analysis of surface KATP channel expression levels requires cell fractionation, protein separation, and quantification. Cell fractionation involves time-consuming high-speed centrifugation steps and skilled techniques for taking out specific layers. Moreover, contamination of intracellular membranes can confound results. Although commercial kits have been developed to make it easier for scientists, qualities of these kits vary which can give rise to variable results. Detection of membrane proteins using surface biotinylation technique consists of labeling cell surface proteins with a biotin reagent before lysing the cells, and isolating these tagged proteins by NeutrAvidin pulldown. Then, the samples are subjected to SDS-PAGE separation, transferred to PVDF membranes, and probed with specific antibodies. Quantification of cell surface expression is accomplished by densitometric measurement of the bands corresponding to the protein of interest and subsequent normalization by a membrane protein (as control). This alternative method for detecting expression of surface protein is relatively easy in steps and more economical in comparison to other methods such as cell fractionation.