Introduction: Hydrocephalus is a neurological disorder characterized by accumulation of cerebrospinal fluid (CSF) within the brain caused by an overproduction of CSF, blockage of flow, or decreased reabsorption. The CSF accumulation causes pathologies including ventriculomegaly, increased intracranial pressure, and cell damage. CSF is secreted by the choroid plexus, a fenestrated capillary network surrounded by an epithelial monolayer in the ventricles. This epithelium is composed of polarized choroid plexus epithelial cells (CPe) in which ion channels are located on either the apical (CSF-facing) or basolateral (blood-facing) membrane. The production and amount of CSF is regulated by a complex interaction between these channels including transient receptor potential vanilloid 4 (TRPV4), calcium-activated potassium channel (KCa3.1), K⁺-Cl^– cotransporter (KCC) and the sodium, potassium 2 chloride channel (NKCC1). TRPV4 is an osmo-, shear-, temperature- and pressure-sensitive nonselective cation channel. TRPV4 activation causes an influx of Ca²⁺ and Na⁺ which can secondarily activate KCa3.1. TRPV4 activation also involves the Ste20/SPS1-related proline-alanine-rich kinase (SPAK) pathway which inhibits KCC and regulates NKCC1. Altering the extracellular K⁺ concentration within physiological parameters also has the potential to affect transport via electrochemical gradients.

Aim: Our goal was to identify a relationship between activation of TRPV4 and potassium channels within the CPe. Identifying the regulation of these channels, will provide further understanding of CSF production in normal and pathophysiological states and provide a basis for identifying potential targets for future drug development for a variety of diseases including hydrocephalus.

Methods: The human choroid plexus papilloma (HIBCPP) cell line was cultured in media supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin, 5 ng/L insulin, and sodium bicarbonate. Cells were seeded onto permeable supports until they formed a monolayer with resistances <400 ohm.cm². Ussing-chamber electrophysiology was used to measure net ion flux and changes in cellular permeability. The relationship between ion channels were investigated using treatments including increasing extracellular K⁺ to 10 mM, 50µM (Dihydroindenyl)oxy alkanoic acid (DIOA, KCC inhibitor), and 5 µM TRAM-34 (Triarylmethane-34, KCa3.1 inhibitor). Compounds were added to either the apical, basolateral, or bilateral sides to determine polarity. After a short (10 min) drug pre-treatment, 15 nM GSK1016790A (TRPV4 agonist) was added to the cells thereby stimulating a complex ion and fluid transepithelial flux. Each treatment had a minimum of six trials. Statistical significance was determined using a paired multiple T-test. 

Results: When KCa3.1 was inhibited on the apical side, both a decrease in net transepithelial ion flux and membrane permeability occurred. Inhibition on the basolateral side resulted in an increased membrane permeability, while inhibition on both sides led to decreased transepithelial ion flux. When KCC was inhibited on the basolateral side, a decrease in both transepithelial ion flux and membrane permeability was observed while inhibition on both sides only showed a decrease in transepithelial ion flux. Increasing the concentration of extracellular K⁺ showed no significant change.

Conclusion: Our results show that there is a relationship between TRPV4 and the potassium channels KCC and KCa3.1, but no significant relationship with physiologically relevant changes in extracellular K⁺.