Wang G and Covarrubias M (2006), Voltage-dependent gating rearrangements in the ...

XB-ART-580

J Gen Physiol 2006 Apr 01;1274:391-400. doi: 10.1085/jgp.200509442.

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Voltage-dependent gating rearrangements in the intracellular T1-T1 interface of a K+ channel.

Wang G , Covarrubias M .

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The intracellular tetramerization domain (T1) of most eukaryotic voltage-gated potassium channels (Kv channels) exists as a "hanging gondola" below the transmembrane regions that directly control activation gating via the electromechanical coupling between the S4 voltage sensor and the main S6 gate. However, much less is known about the putative contribution of the T1 domain to Kv channel gating. This possibility is mechanistically intriguing because the T1-S1 linker connects the T1 domain to the voltage-sensing domain. Previously, we demonstrated that thiol-specific reagents inhibit Kv4.1 channels by reacting in a state-dependent manner with native Zn(2+) site thiolate groups in the T1-T1 interface; therefore, we concluded that the T1-T1 interface is functionally active and not protected by Zn(2+) (Wang, G., M. Shahidullah, C.A. Rocha, C. Strang, P.J. Pfaffinger, and M. Covarrubias. 2005. J. Gen. Physiol. 126:55-69). Here, we co-expressed Kv4.1 channels and auxiliary subunits (KChIP-1 and DPPX-S) to investigate the state and voltage dependence of the accessibility of MTSET to the three interfacial cysteines in the T1 domain. The results showed that the average MTSET modification rate constant (k(MTSET)) is dramatically enhanced in the activated state relative to the resting and inactivated states (approximately 260- and approximately 47-fold, respectively). Crucially, under three separate conditions that produce distinct activation profiles, k(MTSET) is steeply voltage dependent in a manner that is precisely correlated with the peak conductance-voltage relations. These observations strongly suggest that Kv4 channel gating is tightly coupled to voltage-dependent accessibility changes of native T1 cysteines in the intersubunit Zn(2+) site. Furthermore, cross-linking of cysteine pairs across the T1-T1 interface induced substantial inhibition of the channel, which supports the functionally dynamic role of T1 in channel gating. Therefore, we conclude that the complex voltage-dependent gating rearrangements of eukaryotic Kv channels are not limited to the membrane-spanning core but must include the intracellular T1-T1 interface. Oxidative stress in excitable tissues may perturb this interface to modulate Kv4 channel function.

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Species referenced: Xenopus laevis
Genes referenced: dpp6 kcnd1 tbx2

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	Figure 1. The T1 zinc binding site is accessible to internal MTSET in three distinct states. All channels were coexpressed with DPPX-S and KChIP-1. Wild-type (WT), C11xA, and C14xA currents from inside-out patches before (black) and after (red) the application of internal MTSET in the resting (left), activated (center), or inactivated (right) states. The units of current and time are pA and ms, respectively. Top, synchronized application of voltage steps (black line) and MTSET concentration jumps (red bar). The MTSET concentration was 200 μM for the activated state and 400 μM for the resting and inactivated states. The inhibition by MTSET during the test pulse is not apparent because the chemical modification and the resulting inhibition were slow relative to channel gating.
	Figure 2. Gating state–dependent accessibility of the Kv4.1 channel T1–T1 interface to internal MTSET. (A) Time courses of C11xA ternary complex inhibition by internal MTSET in the activated (gray), inactivated (hollow circle), and resting (black) states. Solid lines are best-fit exponential decays with the following second-order rate constants (1/τ[MTSET]): 0.038, 0.366, and 22 mM−1s−1 in the resting, inactivated, and activated states, respectively. The remaining fractional current at steady state was 0.2–0.25 in all states. (B) Bar graph summarizing the second-order rate constants of wild-type and C11xA ternary channel inhibition by MTSET.
	Figure 3. The kMTSET in the activated state against the total number of cysteines in the T1 tetramer. The gray boxes (left) represent the available cysteines in the T1–T1 interface for different mutants. Mutants with one cysteine are variants of C13xA and those with two cysteines are variants of C12xA. The dashed line is the best-fit linear regression with a slope of 1.65 mM−1s−1/Cys.
	Figure 4. MTSET inhibition time courses of the Kv4.1-C11xA ternary complex at different membrane potentials. Solid lines are best-fit exponential decays with second-order rate constants (1/τ[MTSET]) of 0.038, 0.42, 4.66, 11.77, and 21.93 mM−1s−1 at −120, −80, −30, +30, and +80 mV, respectively. MTSET concentrations are 200 μM above −30 mV and 400 μM below +30 mV.
	Figure 5. Voltage dependence of MTSET inhibition rate constants of the Kv4.1-C11xA channel. (A) Inside-out macropatch currents of Kv4.1-C11xA ternary, binary (+DPPX-S only), and ternary + Ni2+ (400 μM, external). Currents were evoked by step depolarizations from −100 mV to command voltages between −100 and +160 mV in 20-mV increments. (B) The kMTSET-Vm (filled) and Gpeak-Vm (hollow) relations for the Kv4.1-C11xA channels depicted in A. The solid lines are the corresponding best-fit fourth-order Boltzmann functions with the following midpoint voltages (V1/2) and slope factors (s): V1/2 = −26 (binary), 14 (ternary), and 59 (ternary + Ni2+); s = 32 (binary), 42 (ternary), and 51 (ternary + Ni2+) mV. The maximal second-order rate constants (mM−1s−1) are 22.32 (binary), 21.13 (ternary), and 29.7 (ternary + Ni2+), respectively. The kMTSET-Vm and Gpeak-Vm relations in all three conditions are strongly correlated. The best-fit linear regression of a plot of all Gpeak-kMTSET pairs has a slope = 1.002 and coefficient = 0.9954.
	Figure 6. Disulfide bond cross-linking in the T1–T1 intersubunit interface inhibits ternary Kv4.1 channels. (A) Stereo view of the residues within the rectangle in B. C110 is from one subunit and C131, C132, and H104 are from the neighboring subunit. A standard color scheme is used to represent the relevant atoms. (B) Structural model of the Kv4 T1 domain. Colored sticks represent the residues that coordinate Zn2+ in the crystal structure, and red lines represent the rest of the T1 protein. Relative to its central axis, the model is tilted to emphasize the angle of view for the residues outlined by the rectangle. (C) The inhibition of Kv4.1 mutants by internally applied Cu/P. The available cysteines are indicated above each graph. Each symbol and corresponding error bars are the mean ± SEM of at least four experiments. (D) Bar graph summarizing the percent inhibition by MTSET (400 μM) before (white) and after treatment with Cu/P (black). The number of experiments is indicated above each bar. After the pretreatment with Cu/P, the differential inhibition of the two mutants by MTSET is statistically significant (P < 0.0001).
	Figure 7. Reversibility of the Cu/P-induced inhibition of ternary Kv4.1 channels. (A) Outward currents evoked by a step depolarization from −100 to +80 mV. The currents were recorded in the inside-out configuration before (black trace) and after (red trace) the application of Cu/P to the intracellular side of the channels, and after exposing the same patch to 10 mM DTT (gray trace). The pH of the intracellular solution was 8.6 throughout the experiment. (B) The time course of the experiment shown in A. The currents in A are averages of ∼10 sweeps. From a total of three independent experiments, 32 ± 4% of the current inhibited by Cu/P was recovered upon the treatment with DTT.
	Figure 8. Coupling between voltage-dependent activation and the putative movements of the T1–T1 interfaces and the post-S6 segments. (A) Structural model of a Kv channel based on the 3D crystal structure of the Kv1.2 channel (Long et al., 2005a). The different colors depict distinct functional domains of the Kv channel: pore domain (P, blue), voltage sensing domain (VS, green; including the S4–S5 linker), and the T1 domain (T1, orange). The S1–T1 linkers (gray) connect the VS domain to the T1 domain. Dashed lines represent the approximate boundaries of the lipid bilayer. A section of the channel at the level of the internal boundary of the lipid bilayer is shown on the righthand side of the model. This view illustrates the fourfold symmetry of the channel oligomer with the S6-tails (blue) sitting just above the T1 domains and the S1–T1 linkers extending from each corner of the oligomer. (B) Two hypothetical pathways that explain the voltage dependence of the cysteine accessibility changes in the T1–T1 interface. The section shown in A (right) was used as a template to develop the schematic model of the T1 domain. The blue cylinders represent the post-S6 segments sitting on the T1–T1 interface when the pore is closed. At rest, the T1–T1 interfaces are buried. The upper pathway assumes VS domain-driven displacements of the T1–T1 interfaces via the S1–T1 linkers, which allow the concerted movement of the S6 helix bundle and the resulting opening of the pore. The lower pathway assumes the quasi-simultaneous displacements of the T1–T1 interfaces and the opening of the main S6 gate. In this case, exposing the T1–T1 interface does not depend on a propagated conformational change mediated by the S1–T1 linker. Instead, a direct interaction between the post-S6 segments and the L4 layers of T1 may drive the T1–T1 displacements as the S6 helix bundl e expands to open the pore. In both pathways, the speculative conformational changes expose the Zn2+ site cysteines in a manner that reflects the increase in conductance resulting from the opening of the main activation gate.

References [+] :

Baker, Three transmembrane conformations and sequence-dependent displacement of the S4 domain in shaker K+ channel gating. 1998, Pubmed