Molecular neuroscienceEndocytic trafficking signals in KCNMB2 regulate surface expression of a large conductance voltage and Ca2+-activated K+ channel
Section snippets
Constructs
Four β2 mutant constructs were created by substituting tyrosine (Y) and/or leucine (L) to alanine (A) by site-directed mutagenesis: mutant 1, L30A, L31A; mutant 2, L225A, L226A; mutant 3, L225A and mutant 4, Y222A+L225A. All constructs were subcloned into the pcDNA3 vector (Invitrogen, Carlsbad, CA, USA). hSlo was tagged with the c-Myc epitope at the N terminus (extracellular). Tagged and untagged hSlo behaved similarly (Meera et al., 1997) and for simplicity, we refer to the
β2 has a dominant expression promoting an intracellular punctuated pattern of hSlo
Specific antibodies recognizing hSlo and auxiliary β2 subunits of MaxiK channel were used to determine their expression patterns in HEK293T cells using confocal microscopy and permeabilized conditions (see Experimental Procedures). Fig. 1 displays typical confocal sections of cells that were transfected with either hSlo (A, B), β2 (C, D) or both (E–J) subunits. As previously shown (Zarei et al., 2001), when cells were only transfected with hSlo (A) cells displayed a rather diffuse expression
Discussion
The main finding of this work is that the β2 subunit that confers the MaxiK α subunit, hSlo, an inactivating phenotype can also regulate its surface expression by removing hSlo from the plasma membrane. Two overlapping endocytic signals proximal to β2 C-terminus play pivotal roles in regulating hSlo surface expression as site-directed mutagenesis within these endocytic sites significantly increased its surface expression. Physiologically, MaxiK channel activation acts as a hyperpolarizing force
Acknowledgments
This work was supported by AHA grant 0230225N, NIH grant GM068855 (M.M.Z.), NIH grants HL54970 (L.T.), HD46510 (E.S.) and MBRS RISE GM065925 (R.J.W.). The authors thank Dr. Ning Zhu for preparing the mutants.
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2016, International Review of NeurobiologyCitation Excerpt :For example, β subunits regulate the effects of voltage and Ca2+ on BK channel opening in the plasma membrane (Dworetzky et al., 1996; McManus et al., 1995; Meera, Wallner, Jiang, & Toro, 1996; Tseng-Crank et al., 1996). Additionally, β subunits can alter the trafficking and pharmacological properties of BK channels (Cox, Toro, Pacheco-Otalora, Garrido-Sanabria, & Zarei, 2014; Shruti et al., 2012; Toro et al., 2006; Zarei et al., 2007). For instance, β4 subunits in brain BK channels confer resistance to IbTX (Meera et al., 2000).
An endoplasmic reticulum trafficking signal regulates surface expression of β4 subunit of a voltage- and Ca<sup>2+</sup>-activated K<sup>+</sup> channel
2014, Brain ResearchCitation Excerpt :Finally, mutating RK (297–298) residues will relief β4 retention and enable correct coassembly with hSlo on the cell surface as indicated by immunocytochemistry and changes in activation time constant (Figs. 2, 6). Previously, we successfully used a DNA molar ratio of 1:2 α to β1 or β2 subunit to study β1–2 subunits surface expressions (Toro et al., 2006; Zarei et al., 2007); however, the same molar ratio of α to β4 showed no detectable β4 surface expression (Figs. 1, 2, 6C–E). In contrast, 1:2 M ratio of the α/β4-M2 subunits generated currents in HEK293T cells that exhibited similar properties as α/β4 currents recorded in Xenopus Oocytes (Brenner et al., 2000).
Palmitoylation of the S0-S1 linker regulates cell surface expression of voltage- and calcium-activated potassium (BK) channels
2010, Journal of Biological ChemistryCitation Excerpt :Co-expression studies have also suggested a role for β-subunits in controlling BK channel cell surface expression. Indeed, β1-subunits have been suggested to decrease surface expression (41), and the β2-subunit also appears to modulate BK surface expression via a similar mechanism (42). Therefore, increasing evidence supports an important role for the S0-S1 linker and surrounding transmembrane domains in controlling BK channel cell surface expression.