Abstract
The dynamic expression of voltage-gated potassium channels (Kvs) at the cell surface is a fundamental factor controlling membrane excitability. In exploring possible mechanisms controlling Kv surface expression, we identified a region in the extracellular linker between the first and second of the six (S1-S6) transmembrane-spanning domains of the Kv1.4 channel, which we hypothesized to be critical for its biogenesis. Using immunofluorescence microscopy, flow cytometry, patch clamp electrophysiology, and mutagenesis, we identified a single threonine residue at position 330 within the Kv1.4 S1-S2 linker that is absolutely required for cell surface expression. Mutation of Thr-330 to an alanine, aspartate, or lysine prevented surface expression. However, surface expression occurred upon co-expression of mutant and wild type Kv1.4 subunits or mutation of Thr-330 to a serine. Mutation of the corresponding residue (Thr-211) in Kv3.1 to alanine also caused intracellular retention, suggesting that the conserved threonine plays a generalized role in surface expression. In support of this idea, sequence comparisons showed conservation of the critical threonine in all Kv families and in organisms across the evolutionary spectrum. Based upon the Kv1.2 crystal structure, further mutagenesis, and the partial restoration of surface expression in an electrostatic T330K bridging mutant, we suggest that Thr-330 hydrogen bonds to equally conserved outer pore residues, which may include a glutamate at position 502 that is also critical for surface expression. We propose that Thr-330 serves to interlock the voltage-sensing and gating domains of adjacent monomers, thereby yielding a structure competent for the surface expression of functional tetramers.
Highlights
As transmitter release and muscle contractility [1,2,3], whereas their malfunction plays a role in diverse disease states [3, 4]
Inspection of the entire S1–S2 linker reveals a region that is juxtaposed to the S1 transmembrane domain, which is highly conserved between Kv channels [17] and which, based on the Kv1.2 crystal structure, may lie in close proximity to extracellular pore mouth residues emerging from the tip of S5 in an adjacent monomer (6 – 8, 21)
Using site-directed mutagenesis, whole-cell patch clamp electrophysiology, flow cytometry, and detailed imaging of eYFP-tagged Kv1.4 expressed in cell lines, we report the identification of a highly conserved threonine residue at the S1/S1–S2 linker interface that is absolutely required for cell surface expression of Kv channels
Summary
Materials—Monoclonal antibodies GM130 and anti-calnexin were purchased from BD Biosciences and anti-HA. from Covance Research Products, Berkeley, CA. A PCR fragment corresponding to mRFP (forward primer: 5Ј-CGT CAG ATC CGC TAG CGG CAC CAT GGC CTC CT-3Ј; Reverse primer: 5Ј-ACC TCC ATA GAA GCT TGA GAA TTC CAC CAC ACT GG-3Ј) was inserted into NheI/HindIII-digested WT eYFP-Kv1.4 using the BD In-fusionTM Dry-Down PCR cloning kit (BD Biosciences) as per the manufacturer’s instructions. Intracellular staining of organelles and total (intracellular ϩ surface) protein detection was performed by fixing cells in 4% paraformaldehyde (as described above), and permeabilizing with 0.5% saponin for 5 min followed by incubation with primary antibodies raised against the. Resuspended and the concentration adjusted to 5 ϫ 107 cells/ml Statistical Analysis—All data are expressed as the mean Ϯ with Dulbecco’s modified Eagle’s medium containing 1% FBS S.E. for n samples. The cells were fixed with 100-l aliquots of 2% (w/v) formaldehyde, 100 l of PBSϪ, and 300 l of PBSϩ
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