The SLC4A protein family regulates the transport of bicarbonate across cell membranes, a process linked to CO 2 hydration during respiration. The SLC4A10 protein (NBCn2/Ncbe) is highly expressed in the choroid plexus epithelium and is thought to be involved in cerebrospinal fluid secretion. Dysregulation of NBCn2 has been associated with conditions featuring elevated intracranial pressure, including brain haemorrhages and stroke. Determining how NBCn2 binds and subsequently transports its substrate into the cell is key to understanding how it regulates the intra- and extracellular pH in the choroid plexus epithelium, as well as the cerebrospinal fluid production. However, due to NBCn2’s high sequence homology to other SLC4A members, achieving subtype-selective inhibition remains challenging, and thus, we have used molecular modelling and biochemical assays to pinpoint specific residues of importance in NBCn2. In order to generate models of NBCn2 at different stages in the transport cycle, AlphaFold2 was employed, using relevant structural information and MSA depth tuning. Molecular dynamics simulations were used to study the structures and the stability of the substrate in the binding sites. We identified key binding-site residues and performed protein-ion fingerprint analysis. Substrate dependence and the order in which the ions bind to the binding site were observed in both pH measurements and simulations. The accelerated weighted histogram (AWH) method was used to map the transport mechanism of NBCn2 between the conformational states and highlighted subtle differences in ion binding. Molecular docking using Glide and subsequent simulations revealed how inhibitors bind and block the substrate from being transported. Our results provide a foundation for guiding the development of new inhibitors specifically for NBCn2, which will be crucial for therapeutic advancements to relieve intracranial pressure in patients.
Desdorf et al. (Sun,) studied this question.