Structural Studies and Drug Discovery to Interrogate the Function of Neuronal SLC4 Transporters

PROJECT SUMMARY Members of the SLC4 bicarbonate transporter family regulate cellular pH in essentially all tissues. Among those, SLC4A7, SLC4A8, and SLC4A10 constitute the subfamily of electroneutral, Na+-dependent transporters that are highly expressed in the brain. Control of cellular pH is not only important for essentially all cellular processes, but it uniquely also directly governs neuronal excitability, making SLC4A7, SLC4A8, and SLC4A10 key regulators of neuronal signaling. Unsurprisingly, mutations of these transporters thus cause a variety of neurological and neurodevelopmental disorders, and, for instance, loss of SLC4A10 function has been found to cause intellectual impairment, anxiety, hyperactivity, and episodic seizures in children. Although these clinical findings and studies in animal models highlight the importance of these transporters in human health and disease, little is known about their molecular mechanisms. Moreover, no tool compounds are currently available to further interrogate the function of SLC4A7, SLC4A8, and SLC4A10, and potentially explore therapeutic avenues in seizure disorders and other illnesses. To address these shortcomings we herein propose a multidisciplinary approach to combine insights from structural and biochemical studies, molecular dynamics simulations, structure-based in silico drug development, and validation of probe activity in (patho)physiologically relevant model systems. Our ultimate goal is to provide key mechanistic insights into SLC4 substrate binding, transport and inhibition, and directly leverage these data in the development of useful SLC4 tool compounds with different subtype-selectivity profiles. We specifically propose three independent aims: (i) Elucidate the different substrate stoichiometries of SLC4A7, SLC4A8, and SLC4A10 using a combination of cryoEM, computational approaches, and cellular transport studies; (ii) develop subtype- selective and nonselective SLC4 tool compounds targeting substrate and allosteric binding sites via structure- based computational drug discovery; and (iii) validate utility of novel probe scaffolds in primary neuronal culture, human neurons derived from induced pluripotent stem cells, and brain slices using substrate uptake assays and electrophysiological recordings. Together, our studies are poised to not only provide direct fundamental insights into SLC4 structure and function, but also generate first-in-class probes with which to explore therapeutic avenues at SLC4 in future studies.