*4.7. Real-Time ATP Efflux Assay*

The ability of the transfected HEK293 cells to release ATP into the culture medium was determined using confluent monolayers as described previously [14,19]. In brief, the medium was removed and replaced with 50 µL efflux buffer, consisting of 11.5 mM HEPES (pH 7.4), 130 mM NaCl, 5 mM MgCl2, 1.5 mM CaCl2, and 11.5 mM glucose. The cells were then incubated for 1 hr at 27 ◦C. Next, 50 µL BactiterGlo reagent (Promega) dissolved in efflux buffer was added to each well. Bioluminescence was subsequently determined in real time in a Flex Station 3 microplate reader (Molecular Devices, San Jose, CA, USA) as detailed previously [14,19]. The real-time ATP efflux assay was run at 27 ◦C for the first 1 h and then at 37 ◦C for 2 hr. The initial low temperature allowed endogenous ecto-nucleotidases to degrade the Abcc6-independent background ATP efflux induced by the medium change.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ijms22136910/s1, Figure S1: Structural models of rABCC6 and hABCC6. Figure S2: Electrostatic potential at the extracellular end of the TMDs for ABCC6 and ABCC1, Figure S3: The LTC<sup>4</sup> binding cavity in bABCC1 and hABCC6, Figure S4: Alignment of ABCC1-6 amino acid sequences. Table S1: List of the ABCC1 and ABCC6 sequences and their corresponding UniProtKB codes used to generate the alignment for the homology models. Table S2: Functional consequences of mutating rAbcc6 R1168 and R1220 and corresponding residues in hABCC6, hABCC1 and hABCC2. Supporting information files: TMD1-NBD1\_Alignment.pdf, TMD2-NBD2\_Alignment.pdf, hABCC6\_inward-facing.pdb, hABCC6\_outward-facing.pdb, rABCC6\_inward-facing.pdb, rABCC6\_outward-facing.pdb.

**Author Contributions:** Conceptualization, K.v.d.W. and V.C.; methodology, K.v.d.W., D.P.T. and V.C.; formal analysis, F.S., V.C. and K.v.d.W.; investigation, F.S., S.D., F.N. and G.C.; writing—original draft preparation, V.C., F.S. and K.v.d.W.; writing—review and editing, F.S., V.C., D.P.T., S.P.C.C. and K.v.d.W.; supervision, K.v.d.W.; project administration, K.v.d.W.; funding acquisition, K.v.d.W., D.P.T. and F.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Institutes of Health, Grant R01AR072695 (K.v.d.W.), U.S. Department of State (Fulbright Visiting Scholar Program), National Research, Development and Innovation Office (OTKA FK131946), Hungarian Academy of Sciences (Bolyai János Fellowship BO/00730/19/8, Mobility grant) and the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation Fund (ÚNKP-2020 New National Excellence Program) to F.S., PXE International to K.v.d.W. and F.S. and the Canadian Institutes of Health Research, Grant MOP 133584, to SPCC. Work in the group of D.P.T. is supported by the Natural Sciences and Engineering and Research Council (Canada) and the Canada Research Chairs Program.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
