Beta1-subunits increase surface expression of a large-conductance Ca2+-activated K+ channel isoform.
Auxiliary (beta) subunits of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels regulate the gating properties of the functional channel complex. Here we show that an avian beta1-subunit also stimulates the trafficking of BK(Ca) channels to the plasma membrane in HEK293T cells and in a native population of developing vertebrate neurons. One C-terminal variant of BK(Ca) alpha-subunits, called the VEDEC isoform after its five last residues, is largely retained in intracellular compartments when it is heterologously expressed in HEK293T cells. A closely related splice variant, called QEERL, shows high levels of constitutive trafficking to the plasma membrane. Co-expression of beta1-subunits with the VEDEC isoform resulted in a large increase in surface BK(Ca) channels as assessed by cell-surface biotinylation assays, whole cell recordings of membrane current, and confocal microscopy in HEK293T cells. Co-expression of beta1-subunits slowed the gating kinetics of BK(Ca) channels, as reported previously. Consistent with this, overexpression of beta1-subunits in a native cell type that expresses intracellular VEDEC channels, embryonic day 9 chick ciliary ganglion neurons, resulted in a significant increase in macroscopic Ca(2+)-activated K(+) current. Both the cytoplasmic N- and C-terminal domains of avian beta1 are able to bind directly to VEDEC and QEERL channels. However, overexpression of the N-terminal domain by itself is sufficient to stimulate trafficking of VEDEC channels to the plasma membrane, whereas overexpression of either the cytoplasmic C-terminal domain or the extracellular loop domain did not affect surface expression of VEDEC.
Kim
Eun Young
EY
Dept. of Biology and Biochemistry, University of Houston, Houston, TX 77204-5001, USA.
Zou
Shengwei
S
Ridgway
Lon D
LD
Dryer
Stuart E
SE
D000704:Analysis of Variance; D019921:Biotinylation / Q000379:methods; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D009154:Mutation / Q000502:physiology; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000502:physiology*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D014162:Transfection / Q000379:methods
D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D021122:Protein Subunits; D049452:Green Fluorescent Proteins