Potassium KCa3.1 channel overexpression deteriorates functionality and availability of channels at the outer cellular membrane

The engineered expression of K⁺ channels has been proposed as a potential treatment for epilepsy due to their exceptional ability to hyperpolarize neurons. A number of rodent models of gene therapy have yielded promising outcomes. However, the prevailing viral delivery methods for transgenes lack external control over expression, which may lead to the overproduction of K⁺ channel subunits and subsequent adverse effects. AAV-based expression of the KCNN4 gene in excitatory neurons has recently been demonstrated to suppress seizures by decreasing neuronal spiking activity. In this study, we examine the effects of overexpression of KCNN4, a gene encoding a pore-forming subunit of KCa3.1 channels, in neurons and HEK293 cells at the cellular and subcellular levels. We employ patch-clamp electrophysiology, immunocytochemistry, and imaging of tagged channel subunits to gain insights into the consequences of KCNN4 overexpression. Our results show that at higher expression levels, the number of channels at the cell membrane decreases, while the engineered expression of the KCa3.1 channel shows a peak in efficiency. Furthermore, our experiments demonstrate that KCNN4 overexpression results in decreased availability of other channels on the membrane and compromised functionality of other channels of the cells. These findings raise an important issue regarding the potential side effects of channel-based gene therapy for neurological disorders. It is critical to consider these side effects in order to successfully translate animal models into clinical trials.