Ion channels, transporters, exchangers, and pumps in neurons or muscle myocytes regulate the formation of action potentials and contractile activity. In endocrine cells, they regulate hormone secretion. Ion channels have key roles even in non-excitable cells, for example, by setting the membrane potential and regulating the influx of Ca2+ into cells. There are hundreds of channels, and most are relatively well characterized. A number, however, remains understudied, which is the focus of RFA-RM-22-024. One focus area of our lab is to investigate the roles of pericytes, specialized cells on the abluminal surface of capillary blood vessels. Pericytes have multiple functions, including forming new blood vessels and regulating blood flow. Pericytes are also sentinels of the innate immune system and directly interact with several types of immune cells by secreting chemokines and cytokines, including IFN-γ, TNF-α, IL-1β, and IL-6. We have developed a mouse model in which mCherry is explicitly expressed in pericytes, which allows isolation of pericytes and enrichment to high purity. We have performed global RNA-seq and focused on the transcriptional profiles of >650 ion channels, exchangers, and pumps expressed in brain pericytes. Transcripts of several Cl- and K+ channels were present. Of note, members of the tweety family (TTYH1, TTYH2, and TTYH3) were amongst the top expressing channels in brain pericytes. This finding was corroborated by public databases, showing that Tthy2 is specifically expressed in pericytes of adult mouse lung vascular and perivascular cells. Tthy2 was initially characterized as swelling-dependent volume-regulated anion channels, but later cryo-EM studies could not identify structural features that are consistent with known characteristics of an anion conduction pore. We hypothesize that Tthy2 might be a component or regulator of a volume-regulated anion channel or that it may have non-channel functions. In Aim 1, we will investigate whether tweety proteins act as components of anion channels in microvascular pericytes. We will record volume-regulated anion currents (VRAC) from primary human brain vascular pericytes and compare data with or without CRISPR knockdown of TTYH2. Experiments are also designed to investigate other types of currents. In Aim 2, we will follow up on preliminary findings suggesting that TTYH2 participates as an immune sentinel. Specifically, we found that TTYH2 may be a negative regulator of the cGAS-STING pathway, which controls the production of IFN-β and IL-6 in response to foreign (e.g., viral) DNA. We will test the physiological function of TTYH2 by examining IRF3 phosphorylation and IFN-β type I IFN and IL-6 production after stimulating the cGAS-STING pathway by treating cells with cGAMP or the STING agonist DMXAA. This multi-PI R03 proposal by Drs. William Coetzee and Stefan Feske bring together their unique expertise to better understand the roles of channels, particularly TTYH2, in vascular function and innate immunity. There are currently no FDA- approved drugs that target ion channels for immunological disorders, and the completion of the proposed studies takes us an essential step in the direction of this missed therapeutic opportunity.

Project Narrative TTYH2, a putative component of a volume-regulated anion channel, remains understudied and is a focus of RFA-RM-22-024. In this proposal, we will study TTHY2 as a putative accessory subunit of on channels in brain microvascular pericytes, and also explore its possible role as a negative regulator of chemokine/cytokine release from pericytes and macrophages. This Pilot Project will investigate an understudied ion channel and will significantly move forward our understanding of tweety proteins in the vascular pericytes and their role in innate immunity.