O2-Sensing in Hypoxic Pulmonary Vasoconstriction - Unravelling the Molecular Mechanisms with Focus on Voltage-Gated Potassium-Channels

• Applicant: Dr. Fenja Knöpp
• Subject Area: Anatomy and Physiology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 452531259

The ventilation and perfusion of the mammalian lung is optimally matched to one another via a physiological mechanism referred to as Hypoxic Pulmonary Vasoconstriction (HPV). An impairment of this vital mechanism can - due to insufficient blood oxygenation and subsequent shortage in systemic oxygen (O2)-delivery - lead to death. In this regard, disturbed HPV is often causative for fatal hypoxemia in intensive care patients, which - according to the latest studies - also seems to apply to COVID-19 patients who require mechanical ventilation.Although an impaired HPV can lead to death, the underlying molecular mechanisms of how hypoxia is initially sensed by the pulmonary vasculature are still not fully elucidated. However, it is beyond dispute that HPV is initiated by a hypoxia-driven inhibition of voltage-gated potassium-channels (Kv-channels) in pulmonary arterial smooth muscle cells (PASMCs). Despite Kv-channels are therefore ideal targets for developing novel strategies to treat disturbed HPV - and thus life-threatening hypoxemia - knowledge about how Kv-channels sense hypoxia is scarce. For that reason, the purpose of this research project is to unravel this pivotal O2-sensing mechanism and potentially identify a novel primary O2-sensor in the pulmonary vasculature. On that score, a Kv-channel that is evidently involved in the initiation of HPV, namely Kv1.5, will be heterologously expressed and its reaction to hypoxia functionally characterized by electrophysiological techniques (Two-Electrode Voltage-Clamp and Patch Clamp). In preliminary experiments, co-expression of Kv1.5 with an auxiliary subunit (Kvβ) was essential for O2-sensitivity of this ion channel - thereby potentially representing a novel primary O2-sensor in the pulmonary circulation. Amino acids within the ion channel´s structure that are responsible for its O2-sensitivity will be determined via site-directed mutagenesis. For validating their role in pulmonary O2-sensing in PASMCs, those amino acids that have been ascertained to be responsible for O2-sensitivity will be analogously mutated in primary PASMCs via CRISPR/Cas9 mediated gene-editing. Finally, the physiological relevance of both, these amino acids as well as the Kvβ-subunits for HPV will be verified in an animal experiment. In summary, the successful elaboration of this project could not only decipher for the first time the molecular mechanisms underlying the hypoxia-induced inhibition of Kv-channels - and thus initiation of HPV - but also identify a novel primary O2-sensor in PASMCs. These investigations would make an important contribution to identify the fundamental molecular principles of oxygen sensing and HPV-initiation - which is a prerequisite for developing novel treatment strategies for disorders which are based on impaired HPV.

• DFG Programme: Research Grants