Early-stage Determinants of T1–S1 Conformations in Kv1.3 Channels

Figures

• 

  Figure 1.. Kv 1.3 channel. Two adjacent subunits of the tetrameric mature Kv1.3 (PDB 7SSX) (left.) The assembled voltage-sensing domain (VSD, S1–S4), the permeation pore (S5–S6), cytosolic tetramerization domain (T1), the T1–S1 linker, and the “window” between T1 and the transmembrane segments (S1–S6) are highlighted. One subunit is colored red, one blue. On the right, a magnification of the T1–S1 linker in one subunit, including the S0 region shown as an α-helix reported in X-ray crystal and cryo-EM structures, and α5, which is the C-terminal segment of the T1 domain. This magnification is reoriented to visualize all portions of the T1–S1 linker (residues 142–183).

• 

  Figure 2.. Accessibility and location of T1–S1 linker residues in monomeric Kv1.3. A. Kv1.3 amino acid sequence for the T1–S1 linker and α5. B. Accessibility using pegylation of cysteine substitution at residues 155 (N-terminal T1–S1 linker), 169 (S0), and 178 (C-terminal T1–S1 linker) in membrane-inserted full-length Kv1.3. Unpegylated and singly-pegylated protein are marked as 0 and 1, respectively. The two intensities at band 0 are core glycosylated (upper) and unglycosylated (lower) protein. C. Fraction of total protein pegylated (Fpeg) (n = 3 mean ± SEM) in the absence of detergent. Lower Fpeg values indicate less accessibility. S0 bars are colored blue. D. Detergent-dependent relative accessibilities. Pegylation of residues 153, 166, and 175 in the absence of detergent, in C12M, or in SDS. Bands as defined in B. E. Relative ratio of Fpeg in C12M to Fpeg in SDS for T1–S1 linker residues (n = 3, mean ± SEM). S0 bars are colored blue.

• 

  Figure 3.. Modulation of linker location by the presence of T1 and T1–T1 Tetramerization. A. Detergent-dependent relative accessibilities for residues in the T1–S1 linker in both WT (solid bar) and T1 (stippled bar). For both constructs, translation was carried out at 22 °C for one hour in the presence of membranes. Data represent means ± SEM, n = 3. B. Fraction of total protein pegylated (Fpeg) for T1–S1 linker residues in both monomer (solid bar) and tetramer (striped bar) (n = 3, mean ± SEM) in the absence of detergent. For the monomer, translation was carried out at 22 °C for one hour in the presence of membranes. For the tetramer, translation was carried out at 30 °C for two hours in the presence of membranes.

• 

  Figure 4.. S0 conformation in nascent chain Kv1.3. A. Sequence of the T1–S1 linker as indicated in Figure 2A including KpnI restriction site and S0 residues mutated to create engineered cysteine pairs 165C/169C, 168C/172C, and 165C/173C. B. Intramolecular crosslinking of ribosome-attached nascent peptides. Left gel: all lanes derived from translations of KpnI-cut mRNA in the absence of membranes (“mm” represents microsomal membranes). Lane 1 shows unmodified protein. Lanes 2–4 show crosslinking of 165C/169C, 168C/172C, and 165C/173C, respectively. Right gel: all lanes derived from membrane-containing translation of 165C/169C (lane 1), 168C/172C (lane 2), and 165C/173C (lane 3). Products were treated sequentially with PDM, DTT, and PEG-MAL. Symbols to the right represent doubly pegylated (F2), singly pegylated (F1) and unpegylated (F0) protein.

• 

  Figure 5.. Specific regions of T1–S1 differentially affect tetramerization. A. Kv1.3 amino acid sequence as indicated in Figure 2A, including residues used to generate ten deletion constructs and two mutation constructs (+PP). B. Quaternary folding of T1 domains. Multimer formation for WT (lane 1) and for T1–S1 deletion constructs (lanes 2–4). Modification with PDM yields monomer, dimer, trimer, and tetramer, indicated by the number of asterisks, respectively. Two cysteines in the T1–T1 interface (118C/126C) were engineered in full-length EcoRI-cut background for this purpose. C. Fraction of total protein crosslinked as dimer, trimer, tetramer divided by the sum of monomer, dimer, trimer, and tetramer (mean ± SEM), i.e., equivalent to the probability of intermolecular crosslinking (Pxlink). D. Fraction of crosslinked protein that is trimer + tetramer, calculated as the sum of trimers + tetramers divided by the sum of dimers, trimers, and tetramers.

• 

  Figure 6.. Core Kv segments lead to a redistribution of multimeric species. A. Sequence of Kv1.3 showing restriction sites. B. Intermolecular crosslinked multimers for the indicated cut-sites KpnI and EcoRI in a 118C/126C Kv1.3 background. Modification with PDM yields monomer, dimer, trimer, and tetramer, indicated by the number of asterisks, respectively. Translation was carried out at the indicated temperatures in the presence of microsomal membranes. C. Fraction of crosslinked protein present as trimer + tetramer as described in Figure 5D.