Slick (Slo2.1), a rapidly-gating sodium-activated potassium channel inhibited by ATP

Abstract

Neuronal stressors such as hypoxia and firing of action potentials at very high frequencies cause intracellular Na+ to rise and ATP to be consumed faster than it can be regenerated. We report the cloning of a gene encoding a K+ channel, Slick, and demonstrate that functionally it is a hybrid between two classes of K+ channels, Na+-activated (KNa) and ATP-sensitive (KATP) K+ channels. The Slick channel is activated by intracellular Na+ and Cl- and is inhibited by intracellular ATP. Slick is widely expressed in the CNS and is detected in heart. We identify a consensus ATP binding site near the C terminus of the channel that is required for ATP and its nonhydrolyzable analogs to reduce open probability. The convergence of Na+, Cl-, and ATP sensitivity in one channel may endow Slick with the ability to integrate multiple indicators of the metabolic state of a cell and to adjust electrical activity appropriately.

Figure 1.

Amino acid sequence alignment of rat Slick (rSlick), human Slick (hSlick), rat Slack (rSlack) and the Slack ortholog from C. elegans, nSlo-2. Sequences were aligned using the web-based program ClustalW from the European Bioinformatics Institute. Residues in gray represent identical amino acids in all four aligned sequences. Gaps are represented by dashed lines. Consensus site for ATP binding to rSlick and hSlick is GXXXXGKT and is boxed. All other designations refer specifically to the rSlick sequence. Transmembrane domains are marked by bars and are designated S1-S6 or P (for pore). Consensus sites are indicated for phosphorylation by protein kinase C (circles) and by cAMP-dependent protein kinase (triangles) and for N-linked glycosylation (ψ).

Figure 2.

Tissue distribution of Slick. A, Northern blot analysis of rat Slick transcripts. A single 6.9 kb band was detected in both brain and heart. B, Slick immunoreactivity in the MNTB and the hippocampus (C). Strong immunolabeling was found in the CA1 section. The antibody was designed against the C-terminal region of Slick and immunohistochemistry was performed using an avidin—biotin-diaminobenzidine reaction system. Scale bars: B, 133 μm; C, 200 μm.

Figure 3.

Expression of Slick and Slack in CHO cells. Whole-cell recordings and associated current-voltage relationships of representative cells transfected with Slick (A) and Slack (B). Recordings were made in bath solutions containing (in mm): 5 NaCl, 140 KCl, 29 glucose, 1 CaCl₂, and 25 HEPES, pH 7.4. The voltage protocol consisted of 200 msec steps from -120 to +120 mV in 20 mV increments from a holding potential of -70 mV. Pipette solution contained (in mm):130 KCl, 5 EGTA, and 10 HEPES, pH 7.2. C, Representative Slick-transfected cell under identical recording conditions as in B except that [Cl^-] was reduced to 5 mm in the pipette solution by replacement with gluconate. D, Cl^- sensitivity of Slick. Average current-density versus voltage of cells recorded in 130 mm Cl^- (n = 5), 30 mm Cl^- (n = 5), and 3 mm Cl^- (n = 4). E, Single-channel properties of Slick and Slack (F) channels. Excised inside-out patches of transfected CHO cells were recorded in a symmetrical solution containing (in mm): 130 KCl, 5 Na-gluconate, 5 EGTA, 10 HEPES, and 29 glucose. All-points histograms for channel recordings are shown in insets. Subconductances for both Slack and Slick were often observed. Recordings were conducted at +80 mV. The “C” and “O” labels represent data from the closed and main open state, respectively. Arrows indicate subconductance states.

Figure 4.

Pharmacology of Slick and Slack. Fractional unblocked whole-cell currents recorded at 0 mV for Slick (A) and Slack (B) using 20 mm tetraethylammonium (TEA), 100 nm iberiotoxin (IbTX), 100 nm charybdotoxin (CTX), 100 nm apamin (Apa), and 1 mm quinidine (Quin). All recordings were performed in a bath consisting of physiological saline and a pipette solution containing (in mm): 130 KCl, 5 EGTA, and 10 HEPES, pH 7.4. C, Voltage-dependent block of Slick by Ba²⁺. The fraction of unblocked current at the end of a 200 msec voltage step in 1 mm Ba²⁺ was measured at 0 and +120 mV (n = 3). Block was greater at the higher voltage. Inset is a recording from a representative cell before and after perfusion of Ba²⁺ at the given voltages. In the presence of Ba²⁺ current decay became stronger at increasingly depolarized potentials. All pharmacological agents were applied extracellularly.

Figure 5.

Slick and Slack are activated by intracellular Na⁺ in inside-out patches from transfected CHO cells. A, Patch recording from a CHO cell transfected with Slick. The cytoplasmic face of the patch was perfused with 0 or 50 mm intracellular Na⁺, as indicated. Membrane potential was held at -80 mV. B, Representative patch recording of inward and outward Slick channel activity in the presence of either 0 or 90 mm Na⁺. Patches were held at 0 mV and stepped to +80 mV for 200 msec and immediately stepped down to -80 mV for another 200 msec. Fifty sweeps were recorded and superimposed onto each other. Basal activity is significant even in the absence of Na⁺. Although the unitary conductance appears inwardly rectifying because of the difference in K⁺ concentrations across the membrane (external [K⁺] = 130 mm, internal [K⁺] = 40 mm), channel openings are favored at positive potentials, consistent with the intrinsic outward rectification of whole-cell currents. C, Dose-response relationship of Na⁺ for Slick. Patches were perfused with solutions containing concentrations of Na⁺ that ranged from 0 to 90 mm, with NMG used as a cationic substitute. [K⁺] was kept constant at 40 mm, and [Cl^-] was kept constant at 30 mm. NP_O values were calculated over 50 sweeps at each dose of Na⁺. NP_O values were then normalized to the NP_O obtained at 90 mm Na⁺. Each point represents the average value from five experiments. Error bars represent SEM. Data was fitted as described in the Materials and Methods section. EC₅₀ was determined to be 89 mm with a Hill coefficient of 1.4. D, Representative patch of Slack channels perfused with Na⁺ as in B. Macroscopic currents were easily measured for Slack, and a time-dependent component of the current was always observed. E, Dose-response relationship of Na⁺ for Slack. Macroscopic currents were measured at each concentration of Na⁺ and normalized to the current elicited at 90 mm. Each point represents the average value from five experiments. Error bars represent SEM. EC₅₀ was determined to be 41 mm with a Hill coefficient of 2.4. For both Slick and Slack channel recordings, outward currents are smaller than inward currents because of the fact that external [K⁺] > internal [K⁺].

Figure 6.

Slick channels are activated by intracellular Cl^-. A, Representative excised inside-out patch recording of Slick channels perfused with increasing concentrations of Cl^-. [K⁺] was 130 mm in perfusate as well as in the pipette solution. Patches were held at 0 mV and stepped to +80 mV for 200 msec. Gluconate was used as a replacement for Cl^-. B, Cl^- dose-response relationship for Slick and Slack. NP_O values were calculated over 50 sweeps at each dose of Cl^-. NP_O values were then normalized to the NP_O obtained at 3 mm [Cl^-] (n = 3 for both Slick and Slack). Error bars represent SEM.

Figure 7.

Intracellular ATP reduces Slick whole-cell currents. A, Average Slick current densities as a function of voltage recorded from transfected CHO cells with a pipette solution containing (in mm): 130 KCl, 5 EGTA, and 10 HEPES, pH 7.2, and 5 MgATP, MgADP, MgATPγS or no nucleotide. Currents were elicited as 200 msec steps to 0 mV from a holding potential of -70 mV. B, Effects of AMP-PNP. The experiment was repeated as in A; however, the nonhydrolyzable ATP analog, AMP-PNP, was used in place of nucleotide in some experiments. These experiments represent a different set of transfected cells versus A. C, Effects of the K_ATP channel agonist diazoxide and the K_ATP antagonist glybenclamide. Recordings were performed as in Figure 4, A and B. Glybenclamide (100 μm) and diazoxide (1 mm) were applied to the bath solution.

Figure 8.

Slick channels are directly inhibited by ATP. Representative excised inside-out patch recordings of Slick channels perfused with a solution containing (in mm): 130 KCl, 5 Na-gluconate, 5 EGTA, 10 HEPES, pH 7.2, and 5 MgATP (A) or 5 mm Na₂ATP (B). Patches were held at 0 mV and stepped to +80 mV for 200 msec. C, Slick channels with mutated consensus ATP binding site (G1032S) perfused with 5 mm MgATP. The activity of the patch remained unchanged after addition of ATP to the bath solution. D, Wild-type Slick channels are inhibited by ATP, but G1032S mutants are not. NP_O values were calculated over 50 sweeps before and after perfusion of patches with ATP. NP_O values were then normalized to the NP_O obtained before perfusion of ATP. Average data are from five experiments for wild-type and four experiments for the G1032S mutant. Error bars represent SEM.