Abstract
Large conductance Ca(2+)-dependent potassium (K(Ca) or maxi K) channels are composed of a pore-forming alpha subunit and an auxiliary beta subunit. We have shown that the brain-specific beta4 subunit modulates the voltage dependence, activation kinetics, and toxin sensitivity of the hSlo channel (Weiger, T. M., Holmqvist, M. H., Levitan, I. B., Clark, F. T., Sprague, S., Huang, W. J., Ge, P., Wang, C., Lawson, D., Jurman, M. E., Glucksmann, M. A., Silos-Santiago, I., DiStefano, P. S., and Curtis, R. (2000) J. Neurosci. 20, 3563-3570). We investigated here the N-linked glycosylation of the beta4 subunit and its effect on the modulation of the hSlo alpha subunit. When expressed alone in HEK293 cells, the beta4 subunit runs as a single molecular weight band on an SDS gel. However, when coexpressed with the hSlo alpha subunit, the beta4 subunit appears as two different molecular weight bands. Enzymatic deglycosylation or mutation of the N-linked glycosylation residues in beta4 converts it to a single lower molecular weight band, even in the presence of the hSlo alpha subunit, suggesting that the beta4 subunit can be present as an immature, core glycosylated form and a mature, highly glycosylated form. Blockage of protein transport from the endoplasmic reticulum to the Golgi compartment with brefeldin A abolishes the mature, highly glycosylated beta4 band. Glycosylation of the beta4 subunit is not required for its binding to the hSlo channel alpha subunit. It also is not necessary for cell membrane targeting of the beta4 subunit, as demonstrated by surface biotinylation experiments. However, the double glycosylation site mutant beta4 (beta4 N53A/N90A) protects the channel less against toxin blockade, as compared with the hSlo channel coexpressed with wild type beta4 subunit. Taken together, these data show that the pore-forming alpha subunit of the hSlo channel promotes N-linked glycosylation of its auxiliary beta4 subunit, and this in turn influences the modulation of the channel by the beta4 subunit.
Highlights
Large conductance Ca2؉-dependent potassium (KCa or maxi K) channels are composed of a pore-forming ␣ subunit and an auxiliary  subunit
We have shown that the brain-specific 4 subunit modulates the voltage dependence, activation kinetics, and toxin sensitivity of the hSlo channel
We found that the highly glycosylated 4 is seen only in cells that express the hSlo ␣ subunit. 4 was coexpressed with a variety of other ion channels and a cytoplasmic dynein light chain, and cell lysates were tested for the presence of the highly glycosylated form of 4
Summary
Cloning and Transient Expression in Chinese Hamster Ovary Cells—4 (GenBankTM accession number AF215891) was cloned into a pcDNA3.1 V5/His Topo vector (Invitrogen) for biochemical investigations. When expressed in Chinese hamster ovary cells, the V5-tagged 4 and untagged 4 modulate hSlo activity. Electrophysiology—For electrophysiological recordings Chinese hamster ovary cells were seeded on glass coverslips and transfected 1–2 days later with hSlo and the appropriate 4 DNA or hSlo and the pIRES2-EGFP vector by using LipofectAMINE 2000 reagent (Invitrogen). V5 epitope-tagged 4 in pcDNA3.1 V5/His Topo was expressed in HEK293 cells, either alone or together with hSlo. Thirty-six h after transfection, the cells were lysed (lysis buffer contained 20 mM Tris-HCl/pH 7.5, 10 mM EDTA/pH 8, 150 mM NaCl, 50 mM KCl, 50 mM NaF, 1% CHAPS, mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, and protease inhibitors) and incubated with an antibody raised against mSlo that recognizes hSlo.. The cells were washed three times with phosphate-buffered saline to remove the biotinylation reagent. Statistical significance was assessed by using the Student’s t test in SigmaPlot software
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
