Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels

Abstract

Ventilation is continuously adjusted by a neural network to maintain blood gases and pH. Acute CO2 and/or pH regulation requires neural feedback from brainstem cells that encode CO2/pH to modulate ventilation, including but not limited to brainstem serotonin (5-HT) neurons. Brainstem 5-HT neurons modulate ventilation and are stimulated by hypercapnic acidosis, the sensitivity of which increases with increasing postnatal age. The proper function of brainstem 5-HT neurons, particularly during post-natal development is critical given that multiple abnormalities in the 5-HT system have been identified in victims of Sudden Infant Death Syndrome. Here, we tested the hypothesis that there are age-dependent increases in expression of pH-sensitive ion channels in brainstem 5-HT neurons, which may underlie their cellular CO2/pH sensitivity. Midline raphe neurons were acutely dissociated from neonatal and mature transgenic SSePet-eGFP rats [which have enhanced green fluorescent protein (eGFP) expression in all 5-HT neurons] and sorted with fluorescence-activated cell sorting (FACS) into 5-HT-enriched and non-5-HT cell pools for subsequent RNA extraction, cDNA library preparation and RNA sequencing. Overlapping differential expression analyses pointed to age-dependent shifts in multiple ion channels, including but not limited to the pH-sensitive potassium ion (K+) channel genes kcnj10 (Kir4.1), kcnj16 (Kir5.1), kcnk1 (TWIK-1), kcnk3 (TASK-1) and kcnk9 (TASK-3). Intracellular contents isolated from single adult eGFP+ 5-HT neurons confirmed gene expression of Kir4.1, Kir5.1 and other K+ channels, but also showed heterogeneity in the expression of multiple genes. 5-HT neuron-enriched cell pools from selected post-natal ages showed increases in Kir4.1, Kir5.1, and TWIK-1, fitting with age-dependent increases in Kir4.1 and Kir5.1 protein expression in raphe tissue samples. Immunofluorescence imaging confirmed Kir5.1 protein was co-localized to brainstem neurons and glia including 5-HT neurons as expected. However, Kir4.1 protein expression was restricted to glia, suggesting that it may not contribute to 5-HT neuron pH sensitivity. Although there are caveats to this approach, the data suggest that pH-sensitive Kir5.1 channels may underlie cellular CO2/pH chemosensitivity in brainstem 5-HT neurons.

Expression of Kir2.1 Inward Rectifying Potassium Channels in Optic Nerve Glia: Evidence for Heteromeric Association with Kir4.1 and Kir5.1

“…Overall, the results are consistent with co-assembly of Kir2.1 and Kir5.1 being a special feature of optic nerve glia. In contrast, Kir2.1/Kir4.1 and Kir4.1/Kir5.1 channels appear to be widely expressed throughout the brain [5,8,12,[14][15][16][17][18][19][20][21] and respectively form strongly rectifying and highly pH sensitive channels [22,23]. Notably, co-assembly of Kir2.1 with Kir5.1 results in the formation of an electrically silent channel [31].…”

Genetic mutation of Kcnj16 identifies Kir5.1‐containing channels as key regulators of acute and chronic pH homeostasis

“…This is an intriguing result given that Kir5.1‐containing Kir channels have not been implicated in hypoxia‐sensing mechanisms within the glomus cells of the carotid bodies or the recently proposed astrocyte‐mediated hypoxia‐sensing mechanisms in the brain (31–33). However, Kir5.1 expression has been detected in the carotid bodies (34) and brainstem astrocytes and glia (6,9), and both have known pH sensitivity (10, 35). Despite the loss of the hypoxic ventilatory response in SS Kcnj16−/− rats, it is possible that the chronic metabolic acidosis leads to hyperventilation at rest through a carotid body‐mediated mechanism given that the blood–brain barrier protects the brain to a large extent during systemic metabolic acidosis (36).…”

“…We previously identified genes that encode pH‐sensitive ion channels in serotonin neurons (6), a subpopulation of central respiratory chemoreceptors with an intrinsic pH sensitivity (7, 8). Among other genes we identified was Kcnj16 , which encodes the inwardly rectifying K + (Kir) 5.1 channel.…”

“…Heterotetrameric Kir channels pass K + currents that contribute to resting membrane potential, and those containing Kir5.1 subunits are inhibited as pH drops within a physiologic range (3, 12, 13). Kir2.1, Kir4.1, and Kir5.1 channels are highly expressed in astrocytes, oligodendrocytes, and select neurons in the brain (6,9,14). These channels are also highly expressed in epithelial cells in the distal nephron (15, 16), where they modulate membrane potential in a pH‐dependent manner and participate in K + recycling, Na + and Cl − resorption, and acid secretion (17).…”

“…Among others, Kir family channels modulate the resting membrane potential and the electrical activity of cells through modulating K + flux, and several Kir family channels have inherent pH sensitivity [reviewed in Putnam et al subunit-containing Kir channels may be ideal candidates to detect pH within the physiologic range within central respiratory chemoreceptors. This intrinsic and physiologic pH sensitivity, along with expression within brainstem neurons shown to contribute to the hypercapnic CO 2 chemoreflex (6,14), has led to the hypothesis that Kir5.1-containing Kir channels play a role in pH regulation, particularly in the ventilatory CO 2 chemoreflex.…”