Tweety proteins: their roles in pericytes and macrophages - 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.