Abstract
Plasma osmolality and blood pressure are regulated by vasopressin released from the posterior pituitary gland by hypothalamic magnocellular neurosecretory cells (MNCs) to promote renal water retention and vasoconstriction. Increased plasma osmolality excites MNCs via activation of an Nterminal variant of the transient receptor potential vanilloid (TRPV)-1 channel. ∆N-TRPV1 is mechanically activated by osmotically-induced membrane shrinkage via microtubules bound to the Cterminus. MNCs also express TRPV4, which is mechanically activated by osmotically induced membrane swelling via the forces acting on microtubules and F-actin. ∆N-TRPV1 and TRPV4 can form mechanosensitive heteromers but it is unknown how the cytoskeleton influences mechanosensitivity of ∆N-TRPV1/TRPV4 heteromers. Therefore, the aim of the project was to test the hypothesis that ∆N-TRPV1 and TRPV4 homomers, and putative ∆N-TRPV1/TRPV4 heteromers mechanotransduction depends on F-actin and microtubules.
HEK293 cells were transfected with equal quantities of ∆N-TRPV1 and/or TRPV4 subunits and treated with either. 2 µM Cytochalasin D (CytD) to destabilise F-actin, 0.5 µM jasplakinolide (JSK) to stabilise F-actin or 5 µM Taxol to stabilise microtubules. Control cells were not treated with drugs. The open probability (NPo), maximum current amplitude and area under the curve (AUC) of transfected drug-treated HEK293 cells was measured using cell-attached patch-clamp recording. Positive (+30 cmH2O) and negative (-30 cmH2O) pressure were applied through the patch pipette to mimic cell-shrink and cell-swell. Results were analysed using two-way repeated measures ANOVA.
∆N-TRPV1 was mechanically activated by positive pressure but not by negative pressure at +60 mV holding potential. CytD, JSK and Taxol did not alter ∆N-TRPV1 mechanical activation under positive pressure. TRPV4 was not mechanically activated by positive pressure or by negative pressure at +60 mV holding potential. Putative ∆N-TRPV1/TRPV4 heteromers were mechanically activated by positive pressure at +60 mV holding potential. CytD and Taxol did not alter putative ∆NTRPV1/TRPV4 mechanical activation under positive pressure. JSK decreased putative ∆NTRPV1/TRPV4 mechanical activation under positive pressure. Taken together, the results indicate that F-actin and microtubules mediate putative ∆NTRPV1/TRPV4 heteromer mechanotransduction.