Understanding the role of GPCR-mediated pH sensitivity in hypercapnic breathing regulation at the retrotrapezoid nucleus

Breathing is essential to life and as such it is a tightly regulated and protected process driven by conscious and reflexive mechanisms. One of the reflexive mechanisms that alters breathing in response to increased arterial CO2/lowered arterial pH is the hypercapnic ventilatory response (HCVR). While work on this reflex was first published more than a century ago, the cellular and molecular mechanisms underlying it are still not fully understood. The central nucleus initiating the HCVR is thought to reside on or near the ventral surface of the medulla but there are a number of nuclei within those anatomical boundaries that have been shown to express some intrinsic CO2/pH sensitivity. One such nucleus, first identified due to its observed anatomical projections to the central respiratory pattern generator, is the retrotrapezoid nucleus (RTN). RTN neurons are intrinsically pH sensitive (i.e., pH sensitivity persists under synaptic blockade and when neurons are dissociated ex vivo) and this sensitivity depends on expression of two distinct proton sensors, GPR4 and TASK-2. However, it is unknown whether both sensors control excitability of the RTN generally or if they are only necessary to provide background excitation at homeostatic arterial/cerebrospinal fluid pH. Additionally, there are no specific antibodies for the native form of either protein that are currently available.
This first section of this work aims to characterize the expression pattern of GPR4 using a knock-in approach to introduce a small epitope tag into the endogenous locus to leverage the highly specific antibodies that exist for those epitopes. We then examine expression of Gpr4 mRNA and protein throughout the mouse brain. The second part of this work focuses on demonstrating that it is the specific pH-sensing capacity of GPR4 that is necessary for a normal HCVR and normal pH activation of the RTN. The individual residues necessary for pH sensing are known for both GPR4 and TASK-2. We again use a knock-in approach to generate animals expressing GPR4 containing two distinct pH-desensitizing histidine mutations. We use these animals to demonstrate that pH-sensitive GPR4 is necessary for a normal HCVR and for normal pH sensitivity of RTN neurons without affecting baseline respiration or neuronal excitability. In an appendix, I present preliminary work using an analogous knock-in strategy to alter the pH sensitivity of TASK-2 and measure effects on the HCVR.
All together, this work describes the expression pattern of GPR4 in the brain for the first time and demonstrates the necessity of pH sensing via GPR4 for manifestation of the HCVR, possibly through its role in mediating the pH sensitivity of RTN neurons.