Human TRPC6 Stable Cell Line - HEK293T

Transient Receptor Potential Canonical 6 (TRPC6) channels represent highly promising therapeutic targets for the treatment of renal, pulmonary, and neurological disorders. Consequently, gaining a comprehensive understanding of their regulatory mechanisms is crucial for the development of novel channel modulators with more precise modes of action. TRPC6 channels are recognized as calcium-permeable, receptor-operated cation channels, activated in response to diacylglycerol (DAG)-a downstream product of the phospholipase C (PLC) signaling pathway. As an endogenous activator of TRPC channels, DAG simultaneously activates protein kinase C (PKC). PKC, in turn, can phosphorylate TRPC6 channels, potentially thereby altering their function. Here, researchers investigated whether five putative PKC phosphorylation sites located within the C-terminus of the TRPC6 channel influence its gating properties. By pharmacologically modulating PKC activity and strategically mutating the aforementioned phosphorylation sites (designed to either block or mimic the phosphorylated state), this study observed alterations in the channel's current kinetics. Furthermore, the "normalized slope conductance"-a metric used to quantify differences in the profile of current-voltage relationships-was correspondingly altered. Notably, despite these manipulations, the magnitude of the maximum induced current density generated by the channel remained unchanged. These findings reveal an "activator-specific" difference in the current kinetics of TRPC6 channels-a difference closely linked to C-terminal amino acid substitutions and PKC-dependent signaling. This discovery suggests that phosphorylation-mediated regulatory mechanisms may play a pivotal role in the fine-tuning of channel activity.

To assess whether the activation or inhibition of PKC affects current density and/or current kinetics, researchers co-incubated wild-type TRPC6-overexpressing HEK293T cells with either the potent PKC activator PMA, or the PKC inhibitors Bisindolylmaleimide I (BIM I) or ceramide (N-acetyl-L-erythro-sphingosine) for 20 minutes at room temperature. Compared to wild-type cells, after incubation with PMA (1 µM) to induce PKC phosphorylation, the maximal current density induced by cis-OptoBI-1 was reduced, whereas the current density induced by cis-OptoDArG remained unaffected (Figure 1A, B, I, J). Relative to cells treated with BIM I and ceramide, the cis-OptoBI-1-induced current density was significantly diminished in the PMA-treated cells. Furthermore, compared to wild-type cells, PKC activation resulted in an accelerated rate of inactivation kinetics induced by OptoBI-1 (Figure 1E). However, relative to wild-type cells, the rates of activation and rapid inactivation kinetics induced by OptoDArG were significantly slowed (Figure 1L, O).

Figure 1. PKC phosphorylation and dephosphorylation alter the current kinetics. (Keck M, et al., 2025)