A Direct Interaction between Cyclodextrins and TASK Channels Decreases the Leak Current in Cerebellar Granule Neurons

Simple Summary Cyclodextrins are cyclic oligosaccharides used to deplete cholesterol from cellular membranes. The effects of methyl-β-cyclodextrin (MβCD) on cellular functions originate principally from reductions in cholesterol levels. In this study, using immunocytochemistry, heterologous expression of K2P channels, and cholesterol-depleting maneuvers, we provide evidence of expression in cultured rat cerebellar granule neurons (CGNs) of TWIK-1 (K2P1), TASK-1 (K2P3), TASK-3 (K2P9), and TRESK (K2P18) channels and their association with lipid rafts using the specific lipids raft markers. In addition, we show a direct blocking with MβCD of TASK-1 and TASK-3 channels as well as for the covalently concatenated heterodimer TASK-1/TASK-3. Abstract Two pore domain potassium channels (K2P) are strongly expressed in the nervous system (CNS), where they play a central role in excitability. These channels give rise to background K+ currents, also known as IKSO (standing-outward potassium current). We detected the expression in primary cultured cerebellar granule neurons (CGNs) of TWIK-1 (K2P1), TASK-1 (K2P3), TASK-3 (K2P9), and TRESK (K2P18) channels by immunocytochemistry and their association with lipid rafts using the specific lipids raft markers flotillin-2 and caveolin-1. At the functional level, methyl-β-cyclodextrin (MβCD, 5 mM) reduced IKSO currents by ~40% in CGN cells. To dissect out this effect, we heterologously expressed the human TWIK-1, TASK-1, TASK-3, and TRESK channels in HEK-293 cells. MβCD directly blocked TASK-1 and TASK-3 channels and the covalently concatenated heterodimer TASK-1/TASK-3 currents. Conversely, MβCD did not affect TWIK-1- and TRESK-mediated K+ currents. On the other hand, the cholesterol-depleting agent filipin III did not affect TASK-1/TASK-3 channels. Together, the results suggest that neuronal background K+ channels are associated to lipid raft environments whilst the functional activity is independent of the cholesterol membrane organization.


Introduction
In excitable cells, K2P channels give rise to background currents, which are open constitutively and voltage independent. In mammals, K2P family is constituted by 15 different members, which also have been found in yeast, plants, zebrafish, nematode, and fruit

Immunocytochemistry
The K2P channels' co-localization in cultured Cerebellar granule neuron was studied by immunofluorescence. Briefly, cultured Cerebellar granule cells were fixed with 1× phosphate-buffered saline (PBS) supplemented with 4% paraformaldehyde (PFA) for 20 min at room temperature and were blocked and permeabilized with 2% BSA in PBS containing 0.1% Triton X-100 for 30 min. Fixed CGN neurons were double-labeled by incubation with goat polyclonal antibodies against TWIK-1, TASK-1, TASK-3, or TRESK (1:100 for sc-11481, sc-32067, sc-11317, and sc-51240; Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit polyclonal antibodies against flotillin-2 (1:100, sc-25507; Santa Cruz) or caveolin-1 (1:100, ab18199; Abcam, Cambridge, MA, USA) overnight at 4 • C. Primary antibody incubation was followed by incubation with an Alexa Fluor ® 488 or Alexa Fluor ® 594 secondary antibody (1:1000 for ab150073 and ab150132; Abcam) for 1 h at room temperature. Controls were carried out with CGN and prepared under identical conditions, with the omission of primary antibody, and no fluorescence signal was detected (data not shown). Co-localization of fluorescent labelling was visualized with a laser scanning confocal microscope (Zeiss LSM-700 microscope, Oberkochen, Germany) using a 40× oil-immersion lens and Photometrics SenSys camera (Photometrics, Tucson, AZ, USA). The images were analyzed with ImageJ (National Institutes of Health, Bethesda, Rockville MD, USA; http://rsb.info.nih.gov/ij/ (accessed on 20 July 2022)). Quantitative co-localization analysis of fluorescence microscopy images was carried out using the co-localization threshold plugin [52] of ImageJ, which uses the threshold algorithm of Costes et al. [53]. All experiments were performed less three times and with independent cell cultures.

HEK-293 Cell Studies
The HEK-293 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). HEK-293 cells were cultured in DMEM-F12 media (Invitrogen) supplemented with 10% FBS (Thermo Fisher) and 1% penicillin and streptomycin. Cells were grown in a humidified incubator at 37 • C and 5% CO 2 .
For the electrophysiological experiments, HEK-293 cells were transfected with cDNAs encoding human TWIK-1 (Mutant K274Q, an active unsumoylated channel) (NM_002245), TASK-1 (NM_002246), TASK-3 (AF212829), TRESK (NM_181840), and the concatenated construct TASK-1/TASK-3 (a covalently linked heterodimer channel [13]). Co-transfections of plasmids containing cDNAs of interest and a reporter vector encoding the cDNA for green fluorescent protein (GFP) (1-2 µg of DNA plasmid) were achieved with a 3:1 ratio (K2P channel plasmid: GFP plasmid) using Xfect polymer (Clontech, Mountain View, CA, USA). The cells were incubated for 3 h in transfection medium OptiMEM (Invitrogen). After incubation, the medium was exchanged with fresh culture medium and maintained at 37 • C with 5% CO 2 for 12 h before electrophysiological measurements were made. All reported studies were performed in at least three independent experiments, with replicate transfections in each experiment.

Electrophysiology
For whole-cell recordings, HEK-293 cells were transfected with the different K2P wild type or chimeric channels, using a PC-501A patch clamp amplifier (Warner Instruments) and borosilicate glass pipettes as previously described by Zúñiga et al. [23]. The cells were superfused with a solution containing 135 mM NaCl, 10 mM HEPES, 10 mM Sucrose, 5 mM KCl, 1 mM MgCl 2 , and 1 mM CaCl 2 and adjusted to pH 7.4 with NaOH. The pipette was filled with a solution of 145 mM KCl, 10 mM HEPES, 5 mM EGTA, and 2 MgCl 2 , pH 7.4 adjusted with KOH. Methyl-β-cyclodextrin (MβCD), α-cyclodextrin (αCD) (Sigma-Aldrich, St. Louis, MO, USA), and filipin III (Cayman chemical, Ann Arbor, MI, USA) were dissolved in water or DMSO to obtain 100 mM and 500 µg/mL stock solutions. Working concentrations of 5 mM MβCD, 5 mM αCD, and 5 µg/mL filipin III were then prepared by diluting stock solutions with the bath solutions obtaining the desired concentrations. Cells were held at −80 mV, then currents were recorded using a protocol of 500 ms of duration from −100 to +100 mV with increments of 10 mV. Patch-clamp acquisition and analysis was conducted with pClamp 10 Software (Molecular Devices). Data analysis was performed using SigmaPlot version 12.0 (Systat Software Inc., San Jose, CA, USA).

Molecular Docking
The TASK-1 crystal structure (Protein Data Bank, PDB: 6RV2) was used for docking calculations. The structure of MβCD was obtained from the crystal structure of gastric inhibitory polypeptide receptor (PDBID 2QKH) that was cocrystallized with MβCD [54]. The MβCD and αCD ligands were designed using LigPrep (Schrödinger, LLC, New York, NY, USA, 2017) force field OPLS-2005, with a maintained charge during the parametrization. Then, the compounds were minimized using Macromodel (Schrodinger, LLC, New York, NY, USA, 2017). We performed molecular dockings using Glide software and the standard precision (SP) scoring function to find the better pose of the CD interacting with TASK-1 structure [55]. For the pose's generation, ten poses were considered per conformer and the strain correction for the GlideScore. Poses were compared and analyzed. The binding sites for MβCD and αCD that shared the most residues in common between TASK-1 and TASK-3 channels were selected.

Statistical Analysis
Data were analyzed with the SPSS software package (SPSS Inc., Chicago, IL, USA). Statistical comparison between groups of data were made using paired Student's t-test. p < 0.05 Values were considered as significant. All data shown are mean ± SEM.

K2P Channels Are Associated with Lipid Rafts
CGNs were used as a model to determine whether K2P channels (TWIK-1, TASK-1, TASK-3, and TRESK) were localized in lipid rafts and the potential regulatory effects due to this localization. To this end, we used immunofluorescence and confocal microscopy to evaluate whether K2P channels co-localize with membrane lipid raft markers flotillin-2 ( Figure 1) and caveolin-1 ( Figure 2). The co-localization of K2P channels with flotillin-2 and caveolin-1 proteins in CGN can be inferred by merging the green and red channels, in the same focal plane, as shown by the yellow color ( Figures 1C,F,I,L and 2C,F,I,L). The correlation between pixel intensity histogram of membrane lipids markers (red channel) and K2P channels (green channel) was analyzed by Pearson's correlation (coefficient values "1" and "0" correspond to perfect co-localization and completely random uncorrelated distribution, respectively) ( Figures 1M and 2M). The Pearson's coefficient (R 2 ) for flotillin-2 and K2P channels TWIK-1, TASK-1, TASK-3, and TRESK is 0.611, 0.850, 0.685, and 0.664, respectively ( Figure 1M), suggesting significant co-localization. Caveolin-1, another specific marker of lipid rafts, showed a significant co-localization with TWIK-1, TASK-1, TASK-3, and TRESK and Pearson's coefficient of 0.758, 0.846, 0.619, and 0.771, respectively ( Figure 2M).

Effect of MβCD on Leak Potassium Currents
To determine whether the presence of K2P channels in lipid rafts plays a regulatory role on the channel function, we disrupted lipid rafts by depleting cholesterol with 5 mM methyl-β-cyclodextrin (MβCD). MβCD treatment reduced the IKSO currents by ~40% at -20 mV in CGNs ( Figure 3A,B). Consistent with this finding, MβCD treatment increased cell input resistance (RIN, p < 0.05) ( Table 1). Conversely, Table 1 shows that the addition of MβCD did not alter the magnitude of the resting membrane potential.
We hypothesized that cholesterol depletion might affect the K2P channels channel gating or activation. Alternatively, the inhibitory effect mediated by MβCD could be explained by a direct interaction between K2P channels and the cyclodextrin. Therefore, we used a heterologous expression system to evaluate the effect mediated by MβCD on each K2P channel (TWIK-1, TASK-1, TASK-3, and TRESK). Double immunostaining analyses revealed a similar extensive distribution and colocalization of TASK-1 channels with the flotillin-2 marker in CGN cells ( Figure 1D), as the dominant channels localized in these domains. Additionally, in a descending order, we found a co-localization of TASK-1 > TASK-3 > TRESK > TWIK-1 ( Figure 1). Similarly, we evaluated the co-localization of K2P channels with caveolin-1 in CGN cells ( Figure 2). Coefficient of determination values (R 2 ) were TASK-1 > TRESK > TWIK-1 > TASK-3 ( Figure 2). Together, the data suggest that at least a fraction of TWIK-1, TASK-1, TASK-3, and TRESK channels are associated with the lipid rafts domain, containing flotillin-2 and caveolin-1 markers, consistent with their presence in lipid raft domains.

Effect of MβCD on Leak Potassium Currents
To determine whether the presence of K2P channels in lipid rafts plays a regulatory role on the channel function, we disrupted lipid rafts by depleting cholesterol with 5 mM methyl-β-cyclodextrin (MβCD). MβCD treatment reduced the IK SO currents by~40% at -20 mV in CGNs ( Figure 3A,B). Consistent with this finding, MβCD treatment increased cell input resistance (R IN , p < 0.05) ( Table 1). Conversely, Table 1 shows that the addition of MβCD did not alter the magnitude of the resting membrane potential. Membrane potential (mV) was determined from current ramps and it is defined as the zero-current potential. The input resistance was obtained according to the Ohm's law from the slope of currents elicited in response to voltage ramps between +10 mV to −10 mV to the resting membrane potential. The values are represented as mean ± SEM. *: indicates statistically significant differences (p < 0.05) by paired t-test.

K2P Channels Sensitivity to MβCD
To dissect the contribution of the K2P channels in the reduction of leak potassium currents in response to MβCD, we independently evaluated the effect of MβCD on hTWIK-1, hTASK-1, hTASK-3, and hTRESK activities in HEK-293 cells transiently transfected with plasmids encoding for the above channels. As shown in Figure 4A-I, MβCD significantly reduced the currents mediated by TASK-1 ( Figure 4E) and TASK-3 channels ( Figure 4G). Conversely, MβCD treatment did not affect TWIK-1 and TRESK-mediated potassium currents ( Figure 4C,I, respectively). K + current reduction observed in TASK-1 and TASK-3 channels could be consistent with an effect of MβCD on the cholesterol distribution or, alternatively, with a direct inhibition of TASK channels by this treatment.  Membrane potential (mV) was determined from current ramps and it is defined as the zero-current potential. The input resistance was obtained according to the Ohm's law from the slope of currents elicited in response to voltage ramps between +10 mV to −10 mV to the resting membrane potential. The values are represented as mean ± SEM. *: indicates statistically significant differences (p < 0.05) by paired t-test.
We hypothesized that cholesterol depletion might affect the K2P channels channel gating or activation. Alternatively, the inhibitory effect mediated by MβCD could be explained by a direct interaction between K2P channels and the cyclodextrin. Therefore, we used a heterologous expression system to evaluate the effect mediated by MβCD on each K2P channel (TWIK-1, TASK-1, TASK-3, and TRESK).

K2P Channels Sensitivity to MβCD
To dissect the contribution of the K2P channels in the reduction of leak potassium currents in response to MβCD, we independently evaluated the effect of MβCD on hTWIK-1, hTASK-1, hTASK-3, and hTRESK activities in HEK-293 cells transiently transfected with plasmids encoding for the above channels. As shown in Figure 4A-I, MβCD significantly reduced the currents mediated by TASK-1 ( Figure 4E) and TASK-3 channels ( Figure 4G). Conversely, MβCD treatment did not affect TWIK-1 and TRESK-mediated potassium currents ( Figure 4C,I, respectively). K + current reduction observed in TASK-1 and TASK-3 channels could be consistent with an effect of MβCD on the cholesterol distribution or, alternatively, with a direct inhibition of TASK channels by this treatment.

TASK-1/TASK-3 Heterodimer Sensitivity to Cyclodextrins and Filipin III
In order to obtain insights into the inhibitory effect mediated by MβCD on TASK-1-and TASK-3-mediated K + currents, we assessed the effect of MβCD on heteromeric TASK-1/TASK-3 channels. This heteromeric configuration, TASK-1/TASK-3, has been studied in native models [13,56] and is a relevant component of IK SO in CGN [13]. As seen in Figure 5B,C, both MβCD and αCD (an inactive cyclodextrin, which does not deplete cholesterol) reduced the potassium currents mediated by TASK-1/TASK-3 concatamers. This effect, mediated by MβCD, is independent of the voltage (Supplementary Figure S1), as the same degree of inhibition was observed in the whole range of voltages studied. To evaluate if the MβCD-mediated inhibition of TASK currents depended on the cholesterol levels, we assessed changes in the activity of TASK-1/TASK-3 concatamers in response to filipin III (5 µg/mL), another cholesterol depleting agent that is not chemically related to MβCD. Filipin III did not have a significant effect on TASK-1/TASK-3-mediated currents, as shown in Figure 5D, suggesting that cholesterol depletion does not affect the K2P channel. Moreover, the application of 3 mM cholesterol did not produce any changes in TASK-1/TASK-3 currents that were significant (n = 4; Supplementary Figure S2).
Biology 2022, 11, x 10 of 16 time course of TWIK-1, TASK-1, TASK-3, and TRESK measured at +60 mV, before and after application of 5 mM MβCD. Results are means ± SEM of at less three different experiments.

TASK-1/TASK-3 Heterodimer Sensitivity to Cyclodextrins and Filipin III
In order to obtain insights into the inhibitory effect mediated by MβCD on TASK-1and TASK-3-mediated K + currents, we assessed the effect of MβCD on heteromeric TASK-1/TASK-3 channels. This heteromeric configuration, TASK-1/TASK-3, has been studied in native models [13,56] and is a relevant component of IKSO in CGN [13]. As seen in Figure  5B,C, both MβCD and αCD (an inactive cyclodextrin, which does not deplete cholesterol) reduced the potassium currents mediated by TASK-1/TASK-3 concatamers. This effect, mediated by MβCD, is independent of the voltage (Supplementary Figure S1), as the same degree of inhibition was observed in the whole range of voltages studied. To evaluate if the MβCD-mediated inhibition of TASK currents depended on the cholesterol levels, we assessed changes in the activity of TASK-1/TASK-3 concatamers in response to filipin III (5 µg/mL), another cholesterol depleting agent that is not chemically related to MβCD. Filipin III did not have a significant effect on TASK-1/TASK-3-mediated currents, as shown in Figure 5D, suggesting that cholesterol depletion does not affect the K2P channel. Moreover, the application of 3 mM cholesterol did not produce any changes in TASK-1/TASK-3 currents that were significant (n = 4; Supplementary Figure S2).

Analysis of Cyclodextrins Binding Sites in TASK-1 Channels
To identify the binding site(s) of the cyclodextrins (MβCD and αCD) in TASK-1 channels, molecular docking was performed in the TASK-1 crystal structure (PDB: 6RV2), which identified eight potential residues located in the extracellular cavity very close to the entry to the TASK-1 channel that might be involved in MβCD ( Figure 6) and αCD (Figure 7) binding: Glu37, Arg68, Lys70, Gly97, Trp184, Gly203, Asp204, and Lys210 (Figures 6D,E and 7D,E).

Analysis of Cyclodextrins Binding Sites in TASK-1 Channels
To identify the binding site(s) of the cyclodextrins (MβCD and αCD) in TASK-1 channels, molecular docking was performed in the TASK-1 crystal structure (PDB: 6RV2), which identified eight potential residues located in the extracellular cavity very close to the entry to the TASK-1 channel that might be involved in MβCD ( Figure 6) and αCD (Figure 7) binding: Glu37, Arg68, Lys70, Gly97, Trp184, Gly203, Asp204, and Lys210 ( Figures 6D,E and 7D,E).      To examine other potential direct interactions between CDs (MβCD and αCD) and TASK-1, we performed molecular docking analysis between CDs in the intracellular cavity of TASK-1 channel ( Figure 8A-C). Four binding potential residues located in the intracellular cavity were found in close proximity to CDs. They are Glu130, Arg245, Glu252, and Lys255 of the TASK-1 channel ( Figure 8D,E). The analyses identified a direct binding involving hydrogen bonds and hydrophobic interactions between CDs and TASK-1 residues.
Biology 2022, 11, x 12 of 16 To examine other potential direct interactions between CDs (MβCD and αCD) and TASK-1, we performed molecular docking analysis between CDs in the intracellular cavity of TASK-1 channel ( Figure 8A-C). Four binding potential residues located in the intracellular cavity were found in close proximity to CDs. They are Glu130, Arg245, Glu252, and Lys255 of the TASK-1 channel ( Figure 8D,E). The analyses identified a direct binding involving hydrogen bonds and hydrophobic interactions between CDs and TASK-1 residues.
Its presence in lipid rafts and cholesterol regulates the activity of several ion channels and plasma membrane proteins, in different ways [46][47][48][49]. Here, we examined the localization of K2P channels in lipid rafts and found that TWIK-1, TASK-1, TASK-3, and TRESK channels co-localized with the lipid raft markers flotillin-2 and caveolin-1. The degree of co-localization varied among the channels, but it is clear that at least part of the K2P channels is localized in lipid rafts. The results of expression in CGNs are in line with previous reports showing that K2P subunits were associated with IKSO currents in CGN cells [13,27]. The expression of TWIK-1, TASK-1, TASK-3, and TRESK in lipid rafts suggests that they may share a common structural core of lipid raft association; however, more work will be required to explore this hypothesis further.
MβCD treatment partially decreased the IKSO current in CGN (~40% Figure 3A,B). This effect was accompanied by an increase in the RIN, which is also consistent with an increased neuronal excitability. The decrease in the amount IKSO current may be related to an effect of disrupting the lipid rafts by depleting cholesterol with 5 mM MβCD [58]. Thus, cholesterol depletion might affect the K2P channel's gating. A similar inhibitory

Discussion
In rat cerebellar granule neurons, TWIK-1, TASK-1, TASK-3, and TRESK subunits forming homodimers and/or heterodimers account for most of the IK SO [13,27]. The IK SO current is critical for modulation of the neuronal excitability and is regulated by several stimuli as muscarinic inhibition, anaesthetics, pH, and sumo/semp activity [13,25,57].
Its presence in lipid rafts and cholesterol regulates the activity of several ion channels and plasma membrane proteins, in different ways [46][47][48][49]. Here, we examined the localization of K2P channels in lipid rafts and found that TWIK-1, TASK-1, TASK-3, and TRESK channels co-localized with the lipid raft markers flotillin-2 and caveolin-1. The degree of co-localization varied among the channels, but it is clear that at least part of the K2P channels is localized in lipid rafts. The results of expression in CGNs are in line with previous reports showing that K2P subunits were associated with IK SO currents in CGN cells [13,27]. The expression of TWIK-1, TASK-1, TASK-3, and TRESK in lipid rafts suggests that they may share a common structural core of lipid raft association; however, more work will be required to explore this hypothesis further.
MβCD treatment partially decreased the IK SO current in CGN (~40% Figure 3A,B). This effect was accompanied by an increase in the R IN , which is also consistent with an increased neuronal excitability. The decrease in the amount IK SO current may be related to an effect of disrupting the lipid rafts by depleting cholesterol with 5 mM MβCD [58]. Thus, cholesterol depletion might affect the K2P channel's gating. A similar inhibitory effect exerted by MβCD has been shown for several ion channels [59][60][61][62][63][64]. In this regard, a previous study has reported a direct inhibitory effect of CDs on K V 1.3 channel, which is independent of membrane cholesterol depletion and concomitant alterations in membrane biophysical parameters caused by CDs [65].
Here, we showed that MβCD decreased TASK-1 or TASK-3 channel activity by~40%. Moreover, no effect of MβCD on TWIK-1 and TRESK channels was observed.
αCD, a cyclodextrin that does not deplete cholesterol from the plasma membrane, showed a similar effect on the tandem dimer channel TASK-1/TASK-3, suggesting a direct effect of cyclodextrins on TASK channels. Another line of evidence that supports the direct effect of cyclodextrin was the treatment with filipin III, which did not show a reduction in TASK-1/TASK-3 mediated currents ( Figure 5D). The lack of effect mediated by cholesterol depletion with filipin III on TASK-1/TASK-3 channels suggests that the amount of cholesterol in the plasma membrane does not play a major role in the regulation of TASK-1 and TASK-3 channels. Moreover, cholesterol enrichment of the plasma membrane did not affect TASK-1/TASK-3 channels (Supplementary Figure S2).
Docking analysis suggested cyclodextrins' binding sites in the extracellular and intracellular cavities of the TASK-1 channel. We propose that the residues involved in the binding site of CD are Glu37, Arg68, Lys70, Gly97, Trp184, Gly203, Asp204, and Lys210 in the extracellular cavity, and Glu130, Arg245, Glu252, and Lys255 in the intracellular cavity.
Extracellular and intracellular binding sites of cyclodextrins in TASK-3 channels are conserved where residues Glu37, Gly97, Trp184, Gly203, Asp204, Glu130, Arg245, and Glu252 might play a key role in cyclodextrin binding. However, it is clear that a mutagenesis approach followed by electrophysiological recordings evaluating the effect of cyclodextrins on mutated TASK-1 and/or TASK-3 channels is certainly needed to discern the role of the residues in the binding sites of CD.
The finding that MβCD blocks the TASK channels in CGN and the heterologous expression system provides evidence and corroborates the role of TASK-1 and TASK-3 channels' activity in the reduction of IK SO . Depolarization that is accompanied by increased R IN is also consistent with increased neuronal excitability.
We suggest that treatment with MβCD on cerebellar granule neurons increases the excitability by a reduction in IK SO , and this effect occurs via a direct interaction with the TASK-1 and TASK-3 channels by a voltage-independent mechanism.

Conclusions
Our study corroborates the localization in lipid rafts of K2P channels and found that TWIK-1, TASK-1, TASK-3, and TRESK channels co-localized with lipid raft markers. The expression of TWIK-1, TASK-1, TASK-3, and TRESK in lipid rafts suggests that they may share a common structural core of lipid raft association. In addition, we show that MβCD treatment decreased the IK SO current in CGN. This effect was accompanied by an increase in the R IN , which is also consistent with an increased neuronal excitability. We suggest that the effect of MβCD treatment on leak potassium currents in CGN cells is by a direct interaction with TASK-1 and TASK-3 channels.

Institutional Review Board Statement:
The animal study protocol was approved the Institutional Bioethics Committee (CIECUAL) of the University of Talca (Code: CIECUAL-UTALCA 22-01).