Regulation of Voltage-gated Potassium (Kv) and Two-Pore Domain Potassium (K2P) Channels implicated in Pulmonary Hypertension

Background

Kv2.1 and TASK-1 channels are two main contributors of K+ currents in pulmonary artery smooth muscle cells (PASMC). Dysregulation of these channels has been implicated in the pathogenesis of pulmonary hypertension (PH). This thesis aims to delve deeper into the implications of the regulation of Kv2.1 by Kv9.3 in PH. Another subject of interest would be whether NADPH oxidase type 4 (Nox4), one of the major reactive oxygen species (ROS) producers in the PASMC, modulates Kv2.1, Kv9.3, and TASK-1 channels. The effects of several redox agents are also investigated as potential modulators of Kv2.1, Kv9.3, and TASK-1. In addition, this thesis also examined the effect of a Kv2-channel blocker, stromatoxin, on Kv2.1 and Kv9.3. Finally, since amphoterin-induced gene and open reading frame (AMIGO) proteins have recently been shown as novel Kv2.1-interacting partners, their effects on Kv2.1 and/or Kv9.3 are also explored in this study.

Experimental approach

Whole-cell patch clamp electrophysiology was used to measure currents of the ion channels expressed in modified tsA-201 cells, in the absence and presence of Nox4 AMIGO and other regulatory molecules. Immunohistochemistry was deployed to visualize the distribution of Kv2.1 and Kv9.3 proteins in the rat lungs and hearts.

Key results and Conclusions

This study supports the findings that Kv9.3 regulates Kv2.1 by increasing the current amplitude, shifting the activation threshold to a more negative voltage range, and prolonging the slow component of time constant of deactivation. These effects could be beneficial in PH as this would mean cells could be brought back to its resting membrane potential faster and the transduction of the next action potential can be delayed. Kv2.1 and Kv9.3 have also been detected at the endothelium and PASMC in rat lungs and hearts, further substantiating the claim that these channels are potential players in regulating PH. AMIGO1 and AMIGO2 proteins are confirmed as regulators of Kv2.1 and Kv9.3 proteins. Nox4 does not regulate Kv2.1, Kv9.3, and TASK-1 channels expressed in tsA-201 cells. While hydrogen peroxide (H2O2) does not have any effect on Kv2.1 and Kv9.3, it abolished the current reduction effect of AMIGO2 on Kv2.1/Kv9.3. Other redox agents used in this study such as dithiothreitol (DTT), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and chloramine T (Ch-T) are not modulators of these channels expressed in tsA-201 cells. The lack of effect from Nox4 and these redox agents could suggest that the redox regulation of different Nox subunit/Kv channels combination varies for different cell types due to the different regulatory proteins present in different heterologous expression systems. As with the case of H2O2 and AMIGO2, it is likely that the regulatory proteins, which could facilitate the hypoxia-sensing properties of Nox4 and the effects of the redox agents on the ion channels, are missing in our heterologous expression system, compared with other host cells.