Identification of Sodium- and Chloride-Sensitive Sites in the Slack Channel

Abstract

The Slack channel (KCNT1, Slo2.2) is a sodium-activated and chloride-activated potassium channel that regulates heart rate and maintains the normal excitability of the nervous system. Despite intense interest in the sodium gating mechanism, a comprehensive investigation to identify the sodium-sensitive and chloride-sensitive sites has been missing. In the present study, we identified two potential sodium-binding sites in the C-terminal domain of the rat Slack channel by conducting electrophysical recordings and systematic mutagenesis of cytosolic acidic residues in the rat Slack channel C terminus. In particular, by taking advantage of the M335A mutant, which results in the opening of the Slack channel in the absence of cytosolic sodium, we found that among the 92 screened negatively charged amino acids, E373 mutants could completely remove sodium sensitivity of the Slack channel. In contrast, several other mutants showed dramatic decreases in sodium sensitivity but did not abolish it altogether. Furthermore, molecular dynamics (MD) simulations performed at the hundreds of nanoseconds timescale revealed one or two sodium ions at the E373 position or an acidic pocket composed of several negatively charged residues. Moreover, the MD simulations predicted possible chloride interaction sites. By screening predicted positively charged residues, we identified R379 as a chloride interaction site. Thus, we conclude that the E373 site and the D863/E865 pocket are two potential sodium-sensitive sites, while R379 is a chloride interaction site in the Slack channel.SIGNIFICANCE STATEMENT The research presented here identified two distinct sodium and one chloride interaction sites located in the intracellular C-terminal domain of the Slack (Slo2.2, KCNT1) channel. Identification of the sites responsible for the sodium and chloride activation of the Slack channel sets its gating property apart from other potassium channels in the BK channel family. This finding sets the stage for future functional and pharmacological studies of this channel.

Keywords: KCNT1 channel; Slo2.2; chloride binding site; gating mechanism; sodium binding site.

Figure 1.

Sodium-sensitive dependence of negatively charged mutants of the rSlack channel. A–C, Typical macroscopic current traces of the WT Slack channel (A), D955N (B), and E490Q (C) mutants were recorded by the inside-out patch configuration. Currents were elicited by a ramp protocol from −100 to +100 mV. D, Sample Hill equation fitted data of the Na⁺ dose–response generated from the macroscopic current of WT [EC₅₀ = 89 ± 3.7 mm, coefficient factor (cf) = 4], E490Q (minimum EC₅₀ = 2716 ± 84.7 mm, cf = 4), and E491Q (minimum EC₅₀ = 1440 ± 64.3 mm, cf = 4) mutants. E, Hill equation fitted data of the Na⁺ dose–response in E787Q (minimum EC₅₀ = 3391 ± 60.3 mm, cf = 4) and E899A (minimum EC₅₀ = 2027 ± 38.4 mm, cf = 4) mutants. F, Hill equation fitted data of the Na⁺ dose response of D955N (minimum EC₅₀ = 1114.3 ± 68.1 mm, cf = 4).

Figure 2.

Summary of Na⁺ sensitivity EC₅₀ values for rSlack mutants. A–D, The sodium dependence EC₅₀ values of 91 mutant constructs (including some double or triple amino acid mutants) are summarized. The Na⁺ sensitivity EC₅₀ values were estimated. *Labels the minimum EC₅₀ values of E490Q, E787Q, and E899Q mutants. The comparison of the EC₅₀ of the WT channel with the EC₅₀ of E490Q (p = 0.0009), E491Q (p = 0.001), E787Q (p = 0.0009), E899Q (p = 0.001), and D955 (p = 0.01) shows significant difference. The mutants (E373Q, D863N, E865Q, and D1196N) that did not show macroscopic currents were labeled with black circles.

Figure 3.

Double mutants with M335A show functional expression but decreased sodium sensitivity. A, Sample single-channel current of M335A mutant with 0 mm intracellular sodium and macroscopic typical current traces with 500 mm cytosolic Na⁺ were recorded in the inside-out patch configuration. A, −100 mV hyperpolarization generated the currents. B, Sample current traces of M335A with 50–500 mm cytosolic Na⁺ are shown. Currents were generated by a −100 to +100 mV ramp protocol. C, Sodium sensitivity of rSlack channel and M335A mutants were estimated by Hill equation fitted data. D, Sample current traces of the M335A/E865Q mutant were recorded in the inside-out patch configuration. E, Hill equation fitted the data of the Na⁺ dose response of D863N/M335A [minimum EC₅₀ = 1154 ± 17.5 mm, coefficient factor (cf) = 4], E865Q/M335A (minimum EC₅₀ = 1430 ± 73.2 mm, cf = 4), and D1196N/M335A (EC₅₀ = 82.6 ± 2.6 mm, cf = 4) mutants. F, Summary of EC₅₀ and minimum EC₅₀ values for the wild-type Slack channel, M335A (EC₅₀ = 49 ± 3 mm, cf = 4), M335A/D863N, M335A/E865Q, and M335A/D1196N.

Figure 4.

Double mutant M335A/E373Q of the Slack channel can be functionally expressed but displays a complete loss of sodium sensitivity. A, Sample single-channel current traces of M335A/E373Q mutant with different cytosolic Na⁺ concentrations. B–D, Total amplitude histogram of single-channel currents with 0−1000 mm [Na⁺]_i was fitted with a Gaussian function. E, Comparison of averaged p_o values of M335A/E373Q in different cytosolic Na⁺ concentrations (Table 3). F, Sample traces of M335A/E373A in 0 and 200 mm [Na⁺]_i, respectively. G, Total amplitude histogram of single-channel currents with 0–400 mm [Na⁺]_i were fitted with a Gaussian function, respectively. The open probabilities of the M335A/E373A in different cytosolic sodium concentrations (Table 3).

Figure 5.

M335A increases the sodium sensitivity of Slack channel mutants. A, Sample current traces recorded in the inside-out patch configuration for Y375A. The ramp protocol ran from −100 to +100 mV. B, Sample current traces recorded in the inside-out patch configuration for the Y375A/M335A mutant with a ramp protocol ran from −100 to +100 mV. C, Hill equation fits the Na⁺ dose dependence data of Y375A and Y375A/M335A mutants. D, Comparison of EC₅₀ values of the sodium dependence of L371A to Y375A with the EC₅₀ values of the sodium dependence of double mutants of M335A plus corresponding mutants from L371A to Y375A. E, Hill equation fits the Na⁺ dose dependence data of M335A/D955N and M335A/E491Q. F, Comparison of minimum EC₅₀ values of sodium dependence of E490Q, E491Q, E787Q, E899A, and D955N with the minimum EC₅₀ values of sodium dependence of M335A/E490Q, M335A/E491Q, M335A/E787Q, M335A/E899A, and M335A/D955N.

Figure 6.

The E373Q mutant is expressed on the cell membrane in transfected HEK293 cells. A, Green fluorescence showing the expression of WT rSlack-EGFP in rSlack-EGFPN1 transfected HEK293 cells. B, The WGA staining on the cell membrane of the fixed HEK293 cells (red). C, The merged image shows the colocalization of WT rSlack-EGFP and WGA staining on the cell membrane of the HEK293 cells. D–F, The magnified picture of one cell shows the colocalization of the EGFP and WGA staining on the cell membrane. G–I, Green fluorescence shows the rSlackE373Q-EGFP expression in transfected HEK293 cells (G), while WGA stains show rSlackE373Q-EGFP expression in the HEK293 plasma membrane (H), and the merged expression of rSlackE373Q-EGFP and WGA in the transfected HEK293 cells (I). J–L, The magnified picture of merged fluorescence signals of the expression of E373Q-EGFP mutant and WGA staining on the plasma membrane in the HEK293 cells. M–R, The EGFP expression and WGA staining image of the R373A mutant. S, The overlap rates of EGFP and WGA staining of the WT, E373Q, and E373A mutants of the Slack channel.

Figure 7.

Western blot (WB) analysis of WT Slack channel, E373Q, and E373A membrane expression in transfected HEK293 cells. A, Western blot results of the expression of WT Slack channel, E373Q, and E373A mutants in total proteins. B, Western blot results of the expression of WT Slack channel, E373Q, and E373A mutants in the membrane protein fractions. C, Alignment of the context segments around the E373 sodium-sensitive site of Slack channel and Slo1 channel in different species. The species protein sequence IDs are as follows: mSlo1 human Slack, XP_011517182.1; rSlack, NP_068625.1; dog Slack, XP_005625191.1; cattle Slack, XP_027412510.1; zebra finch Slack, XP_027412510.1; Drosophila Slo2, EDW47242.1; nSLo2, NP_001024527.1; rat Slack, Q6UVM4.1; and goldfish Slack, XP_026052475.1. D, The alignment of the context segments around the D863/E865 sodium-sensitive site of the Slack channel.

Figure 8.

M335A/E373A and M335A/E373Q mutants show similar single-channel characteristics with the WT rSlack channel. A, The sample traces of M335A mutant in the presence of different cytosolic sodium ions at −100 mV membrane potential. B, The Hill equation fitted the Slack channel open probability versus sodium concentration data. The EC₅₀ value is 46.5 ± 2.3 mm. C, The sample single-channel traces of M335A/E373Q mutant at indicated voltages in intracellular solution with 0 mm cytosolic Na⁺. D, The plotted traces show M335A single-channel conductance is 170 ± 5 pS while M335A/E373Q and M335A/E373A single-channel conductance is 163 ± 10 and 167 ± 4 pS at 0 mm cytosolic sodium, respectively. E, The sample single-channel traces of the M335A mutant were blocked by bepridil at indicated concentrations. F, The Hill equation fitted the dose–response curve of the bepridil blocking on M335A, M335A/E373A, and M335A/E373Q mutants. The blocking EC₅₀ values of bepridil on these mutants are 1.55 ± 0.17, 1.2 ± 0.03, and 1.18 ± 0.04 μm, respectively.

Figure 9.

Molecular simulation shows two potential sodium binding sites in the C terminus of the Slack channel. A, The rmsD of the backbone of the Slack channel during MD simulation. B, The rmsF of total Na⁺ ions in the simulation system of a Slack channel homology model; the red-labeled sodium ions are in the E373 residue, while the green-labeled sodium ions are in the D863 position. C, The distances from the residing sodium ions to residues E373 and D955 in subunit b during the MD simulation process. D, A snapshot of the E373 sodium interaction site with only one sodium ion located between the E373 and D955 residues. E, During the MD simulation, the distances from two simultaneously resident sodium ions to residues E373 and D955 in the subunit c of the Slack channel. F, A snapshot of the simulated local structure of equilibrated coordinated sites with a conformation that two sodium ions simultaneously bind close to the E373 and D955 residues. G, The sodium ion distances to residues D863, E865, E787, E899, E490, and E491 in the acidic sodium pocket during the MD simulation in the Slack channel model. H, Simulated local structure of the acidic pocket formed by E490, E491, E787, D818, D863, and E865 amino acids of the Slack channel.

Figure 10.

The time of the resident sodium ions staying in the sodium-sensitive site of the rSlack channel during the MD simulation. A, The time of four subunits with one or two sodium sites occupied or vacant in the homology model during the 500 ns MD simulation was indicated by different colors. B, The time of the four subunits with one or two sodium sites bound by one or two sodium ions and one vacant site during the 350 ns MD simulation. C, D, The rmsD of the two sodium ions (Na1 and Na2) binding to the E373/D955 site during the 500 ns MD simulation. E, The distance between the E373 and D955 site of one subunit during the 500 ns MD simulation. F, The snapshots of the local structure of the E373/D955 site with (gray) or without (red) sodium binding.

Figure 11.

The location of the sodium binding site on the RCK1 and RCK2 interface in the Slack channel. A, Distances from residue D863 to residues E899, E787, E490, and E491, respectively, during a 500 ns MD simulation. B, The local structure of the sodium binding pocket around D863/E865. The blue color shows residues being closer to residue D863 at 210 ns. The red color shows residues relatively far away from D863 at 140 ns when the site is vacant. C, The rmsD of the backbone of the Slack channel model using 5U76 as the template. D, The rmsF of Na⁺ ions in the Slack channel homology model using the structure of the chSlack channel (PDBID:5U76). The red-labeled sodium ions resided in the D863/E865 pocket, while the green-labeled sodium ions resided in the E373 position. E, Distances from the resident sodium ion to residues E373 and D955 in subunit 1 during the MD simulation. F, The local structure of the sodium ion (Na1) resides in the E373/D955 position.

Figure 12.

The distances from sodium ions to the interacting residues and the snapshot of the local structure of sodium interaction sites. A, The distances from the resident sodium ions (top, Na2; bottom, Na3) to the residues D863/E865/E787/D818 in subunit 1. B, The snapshot of the local structure when two sodium ions resided in the D863/E865 pocket. C, The distances from the Na4 (top) and Na5 (bottom) to D863/E865/E787/D818 residues in subunit 1. D, The snapshot of the local structure D863/E865 pocket when four sodium ions are located in the site. E, The distances from the sodium ions (Na6 top and Na7 bottom) to the residues D863/E865/E787/D818 in subunit 4. F, The local structure of the D863/E865 pocket in subunit 4 with two resident sodium ions.

Figure 13.

Molecular simulation shows that the D863/E865 and E373 mutated channels can no longer bind sodium. A, The rmsD of the backbone of the mutated Slack channel during MD simulation (5U76 as a template). B, The rmsF of total Na⁺ ions in the simulation system of a Slack channel homology model. C, During the MD simulation process, the distances from a sodium ion to residues D863/E865/E787/D818 pocket in subunit b. D, E, Snapshots of the simulated local structure of N863/Q865 (D) and the E373 (E) residues.

Figure 14.

The simulation predicted the Cl^–-sensitive site candidates in the Slack channel. A, The root-mean-square fluctuations (RMSF) of total Cl- ions in the system during the 500 ns MD simulation. B, The predicted amino acid candidate of the Cl- sensitive site of the Slack channel. C, The sequence alignment between the rSlack channel and C. elegans Slo2 channel. The candidate amino acids as the Cl⁻-sensitive site were labeled red.

Figure 15.

The R379Q mutant removes the chloride sensitivity of the Slack channel. A, Typical current traces of K1098Q were recorded in the inside-out configuration perfused with 0–500 mm NaGlu and 0–500 mm NaCl, respectively. B, Hill equation fitted data of the NaGlu (EC₅₀ = 113.5 ± 8.3 mm, n = 3.9) and the NaCl (EC₅₀ = 119.6 ± 7.6 mm, n = 3.8) dose response of K1098Q mutant. C, Typical currents of R379Q were recorded in an inside-out configuration bathed in 0–500 mm NaGlu and NaCl, respectively. D, Hill equation fitted data of the NaGlu (EC₅₀ = 119.1 ± 5.9 mm, n = 3.9) and NaCl (EC₅₀ = 79.4 ± 5.8 mm, n = 3.8) dose response of R379Q mutant, respectively. E, Comparison of candidate mutants of Cl^–-sensitive sites of EC₅₀ values of NaGlu and NaCl dose dependence. EC₅₀ values of all mutants with NaGlu are as follows (in mm): WT, EC₅₀ = 91.6 ± 2.7; R379Q, EC₅₀ = 113.5 ± 8.3; R474Q, EC₅₀ = 220.8 ± 6.6; R626/627Q, EC₅₀ = 231.7 ± 7.5; R912A, EC₅₀ =76 ± 6.1; K914Q, EC₅₀ = 116.8 ± 3.0; K923Q, EC₅₀ = 193.0 ± 18; R947Q, EC₅₀ = 193.6 ± 13; R1093Q, EC₅₀ = 87.1 ± 2.0; R1094Q, EC₅₀ = 104.3 ± 7.0; R1097Q, EC₅₀ = 134.9 ± 6.0; and K1098Q, EC₅₀ = 119.1 ± 5.9. EC₅₀ values of all mutants in NaCl solution are as follows (in mm): WT, EC₅₀ = 71.6 ± 5.1; R379Q, EC₅₀ = 119.6 ± 7.6; R474Q, EC₅₀ = 151.6 ± 6.1; R626/627Q EC₅₀ = 125.2 ± 11; R912A, EC₅₀ = 46 ± 3.8; K914Q, EC₅₀ = 70.7 ± 1.9; K923Q, EC₅₀ = 152.2 ± 3.5; R947Q, EC₅₀ = 130.8 ± 7.4; R1093Q, EC₅₀ = 68 ± 5.6; R1094Q, EC₅₀ = 71 ± 3.8; R1097Q, EC₅₀ = 64.6 ± 5.6; and K1098Q, EC₅₀ = 79.4 ± 5.8.

Figure 16.

Distances of Cl^– to positively charged amino acids R379Q and R626/627Q of rSlack channel in the simulated local structural model. A, Sample traces of R379A in NaGlu and NaCl solution. B, Comparison of sodium sensitivity of the R379 mutant EC₅₀ values in NaGlu and NaCl solution. The sodium dependence of the R379 mutant EC₅₀ value in NaGlu are as follows (in mm): R379A, EC₅₀ = 95 ± 2; R379N, EC₅₀ = 89 ± 2.1; R379C, EC₅₀ = 101.3 ± 4.2; and R379T, EC₅₀ = 99.3 ± 3.3. The EC₅₀ values in NaCl are as follows (in mm): R379A, EC₅₀ = 94 ± 1.6; R379N, EC₅₀ = 85.4 ± 4.4; R379C, EC₅₀ = 104 ± 7.3; and R379T, EC₅₀ = 102.8 ± 4.2. C, During the simulation process, distances of the bound Cl^– ion to residues R379, R626, and R627 amino acids. D, Distances of another bound Cl^– ion to residues R379, R626, and R627 amino acids during the simulation process. E, F, The time course of the current change of the WT Slack channel and R379Q mutant were perfused in different Cl^– and 250 mm sodium solution concentrations. G, The Cl^– dose-dependent curve for the activation of the Slack channel.

Figure 17.

The single channel recording of Cl- dependence of the WT Slack and R379Q mutation. A-B, The sample traces of single channel recording traces of the WT Slack channel (A) and the R379Q mutant (B) under the 0 Cl, 25 Cl, and 50 Cl (in mM). C-F, The histogram analysis of the Po of the WT channel (C and E) and R379Q mutant (D and F), fitted by a Gaussian function. G, Comparison of the Po of the WT channel and the R379Q mutant under 100 Na and 0 Cl (WT, 0.158 ± 0.011; R379Q, 0.585 ± 0.022. t-test, p < 0.05, n = 5), 25 Cl (WT, 0.325 ± 00.046; R379Q, 0.604 ± 0.017. t-test, p < 0.05, n = 5), 50 Cl (WT, 0.489 ± 0.051; R379Q, 0.57 ± 0.076). H, The dose response curve of Cl⁻ with 100 mM intracellular sodium. The Cl⁻ EC₅₀ = 23.74 ± 2.1 mM.

Figure 18.

The sodium-sensitive and Cl^–-sensitive sites are located in the same subunit in the RCK1 and RCK2 domain interface. A, The E373 and D863/E865 sensitive sites are located on the interface of RCK1 and RCK2 domains in the model of the Slack channel. Blue indicates the RCK1 domain, while red indicates the RCK2 domain. The green labels the pore domain, while the yellow labels the transmembrane domain. B, The linear scheme of the Slack channel with the location of the sodium-sensitive and chloride-sensitive sites. The local structure of the RCK1 and RCK2 domains. C, The cartoon structure of the Slack channel shows the location of the sodium-sensitive and Cl^–-sensitive sites.