Mechanically-Induced Gating in PKD2L1 (Trpp2): Calcium-Induced Activation Masquerading as Force Sensitivity?

Abstract

The polycystic kidney disease 2-like protein (PKD2L1) is a member of the TRP channel superfamily and is highly expressed in the developing fetus, especially in the cardiovascular system. In complex with PKD1L1 it forms a complex involved in Ca2+-signaling in primary cilia. Given the ubiquitous expression of PKD2L1 and its localization within primary cilia, long thought of as mechanosensing organelles, we sought to robustly address its mechanosensitivity alone and in the presence of putative binding partners including PKD1L1, TRPC1/6, TRPV4, Piezo1 and PKD1/2. In order to do so we created a stable PKD2L1 expressing doxycycline inducible Flp-In HEK293 cell line. In the cell-attached configuration we could elicit large robust mechanically-activated PKD2L1 currents over pressure ranges largely overlapping with Piezo1. However, in comparison to the mechanically-gated channel Piezo1 whose response was instantaneous to applied force the PKD2L1 channels had a much longer latency period. Once this period was overcome the channel responded to subsequent mechanical stimuli like a bona fide mechanosensitive channel. The activity in cell-attached patches was highly dependent on the presence of divalent cations. PKD2L1 is known to activate in response to rises in intracellular Ca2+ and then subsequently desensitize. We found that multiple responses to mechanical stimuli was only possible in the absence of Ca2+ (n=45) in the pipette. When 1 mM Ca2+ or Ba2+ were present in the pipette current rapidly and irreversibly desensitized. Activity could still be elicited in the presence of BAPTA or EGTA in the extracellular solution indicating that the response was not mediated by external Ca2+ influx. The activity was boosted by the addition of cytochalasin D (10 µM)(n=11) or GsMTx-4 (5 µM)(n=7) and inhibited by colchicine (10 µM)(n=9). This data suggests that PKD2L1 channel activity can be modulated by mechanical force but the exact mechanism still remains to be elucidated.