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Am J Physiol Cell Physiol
2024 Aug 01;3273:C790-C797. doi: 10.1152/ajpcell.00422.2024.
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A positively charged residue at the Kv1.1 T1 interface is critical for voltage-dependent activation and gating kinetics.
Hasan SM
,
Aswad N
,
Al-Sabah S
.
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Within the tetramerization domain (T1) of most voltage-gated potassium channels (Kv) are highly conserved charged residues that line the T1-T1 interface. We investigated the Kv1.1 residue R86 located at the narrowest region of the T1 interface. A Kv1.1 R86Q mutation was reported in a child diagnosed with lower limb dyskinesia [1]. The child did not present with episodic ataxia 1 (EA1) symptoms typically associated with Kv1.1 loss-of-function mutations. We characterized the electrophysiological outcome of the R86Q substitution by expressing Kv1.1 in Xenopus laevis oocytes. Mutated α-subunits were able to form functional channels that pass delayed rectifier currents. Oocytes that expressed only mutated α-subunits produced a significant reduction in Kv1.1 current and showed a positive shift in voltage-dependence of activation. In addition, there was substantially slower activation and faster deactivation implying a reduction in the time the channel is in its open state. Oocytes co-injected with both mutated and wild-type cRNA in equal amounts, to mimic the heterozygous condition of the disease, showed a decrease in current amplitude at -10mV and faster deactivation kinetics when compared to the wild-type channel. These findings indicate that T1 plays a role in Kv1.1's voltage-dependent activation and in its kinetics of activation and deactivation.
Figure 1. (A) Schematic Kv1.1 model showing two of the four α-subunits with the approximate location of residue R86 (red) within the T1 domain of the N-terminus. A K + ion is shown in the transmembrane conduction pore. The T1 pore lies beneath the transmembrane pore with R86 at the T1-T1 interface. (B) Sequence alignment of a portion of the T1 region. The Arginine residue of interest is shown in red. The grey highlighted area is the most conserved portion of the T1 sequence. Kv1.1 R86 is conserved within the Kv1 family of Homo sapiens and in various species. Kv1.1 R47 and D90 (in black bold) are also conserved. The number of the first amino acid in each sequence is indicated. Sequence alignments were generated using BLAST (NCBI, US).
Figure 2. (A) Representative Kv1.1 current recordings from oocytes injected with WT, mutated (R86Q), and WT and mutated (WT/R86Q) cRNA. Voltage-clamp protocol for Kv1.1 currents is indicated on top of the R86Q recording. Oocytes were depolarized to 17 potentials, from -80 to +80 mV from a holding potential of -80 mV. (B)373Representative currents at -10 mV and +80 mV. (C) Left box plot showing significant difference in mean steady-state amplitude between all three groups at -10 mV test potential. The right box plot shows mean current amplitudes for the three groups recorded at +80 mV. Solid lines in box indicate the median, boxes indicate lower and upper quartiles, and whiskers the minimum and maximum. Steady state mean amplitudes were obtained at approximately 400 ms. WT: n=27, R86Q: n=28, WT/R86Q: n=20. *p<0.05, ***p <0.001, **** p<0.0001.
Figure 3. (A) Representative activation tail currents. Protocol for tail currents is indicated above the WT current. (B) Po curves were derived from peak tail amplitudes measured at -40 mV that were normalized to maximum peak and plotted as a function of various pre-pulse potentials. (C) Inactivation curves were obtained from normalized steady state +40 mV currents after various depolarizing 20 s pre-pulses from -80 to +20 mV. Normalized amplitudes were plotted as a function of pre-pulse potentials. Both activation (B) and inactivation curves (C) were fitted with the Boltzmann function.
Figure 4. (A) To the left are the superimposed activation currents recorded at +80 mV. To the right are representative deactivation currents recorded at -20 mV. Currents were normalized to their respective peaks. (B) Activation and deactivation time constants plotted as a function of the test voltage. On the right of the graph are the activation time constants (τ) derived from rising phases of currents. Curves to the left are deactivation curves. Data points represented are mean ± SE, n=8 for each group
Figure 5. (A) Ribbon representation of the side view of the Kv1.1 tetrameric channel showing the location of R86 as a ball-and-stick representation in magenta. (B) View of the Kv1.1 tetramer from the intracellular side showing R86 at the interface in magenta. The potassium ion (yellow sphere) can be seen in the center of the pore. (C) Molecular surface representation of the N-terminal tetramer showing R86 at the T1 cavity interface. Top figure: View from393the intracellular side. Bottom figure: Side view with one N-terminal subunit removed for clarity. (D) Molecular surface representation of four mutated subunits assembled. Top figure: Q86 is not visible from the intracellular side. Arrow indicates the cavity where R86 used to be. Bottom figure: Q86 (red) is only visible from the side view following the removal of one subunit. The mutation resulted in a change to the structure of the T1 cavity. The representation and angle are the same as shown in c. (E) Tetrameric assembly of the T1 domains made up of two WT T1 subunits with R86 represented as ball and sticks in magenta, and two mutated T1 subunits with the Q86 residue in red. This tetrameric two by two configuration is one of several possible combinations of WT and mutated subunit associations View is from the intracellular side.
Figure 6. (A) N-terminal tetramer showing the proximity of D90 (yellow) and R47 (orange) to R86 (magenta) and Q86 (red). (B) Distances between two atoms are indicated as dashes. Numbers are the distance in angstroms of the polar bonds measured using PyMol.
T1 domain plays a role in Kv1.1 gating kinetics and voltage-dependance
