Human TRPC6 Stable Cell Line-HEK293

The actin cytoskeleton of podocytes plays a central role in the function of the renal filtration barrier. Calcium influx into podocytes via TRPC6 (transient receptor potential canonical 6) channels triggers actin cytoskeleton rearrangement, thereby affecting the filtration barrier. Researchers hypothesized the existence of a feedback mechanism wherein the cytoskeleton regulates TRPC6 channel activity. Experiments using scanning ion conductance microscopy demonstrated altered migration characteristics in podocyte cultures treated with cytochalasin D, a drug that disrupts the actin cytoskeleton. Cell-attached patch-clamp recordings revealed that cytochalasin D enhanced TRPC6 channel activity in TRPC6-overexpressing CHO (Chinese hamster ovary) cells and in podocytes within freshly isolated glomeruli. Furthermore, previous studies have shown that mutations in the ACTN4 gene-which encodes α-actinin-4-cause focal segmental glomerulosclerosis and lead to the stiffening of the actin network within podocytes. Consequently, researchers investigated whether α-actinin-4 regulates TRPC6 channel activity. The results showed that co-expression of the mutant α-actinin-4 K255E with TRPC6 in CHO cells reduced TRPC6 channel activity. Collectively, these data confirm a direct interaction between the structure of the actin cytoskeleton and TRPC6 activity.

To investigate how the disruption of actin cytoskeleton dynamics regulates TRPC6, researchers examined the effect of cytochalasin D on TRPC6 channel activity. Several members of the TRPC family-including TRPC3, TRPC5, and TRPC6-have been implicated in the pathogenesis of proteinuric kidney disease. Therefore, to explore the role of the actin cytoskeleton in TRPC6 channel function, the researchers generated TRPC6-overexpressing CHO cells. In the cell-attached recording configuration, the addition of 10 µM cytochalasin D to the bath solution induced single-channel activation (Figure 1a), with a current-voltage relationship characteristic of TRPC6 (Figure 1b). The addition of cytochalasin D did not alter the conductance of the recorded channels. The positive reversal potential observed in the current-voltage curve reflected the cell's plasma membrane potential. Upon excision of the membrane patch to the inside-out configuration, the reversal potential shifted to 0 mV, and the conductance increased to 23 ± 1 pS (n = 6). Control cells transfected only with GFP exhibited no TRPC6 activity either before or after the addition of cytochalasin D. Following the addition of cytochalasin D, the basal activity (NPo) of the overexpressed TRPC6 channels increased (Figure 1c). In experimental groups that did not initially exhibit basal activity, 10 µM cytochalasin D was still able to activate TRPC6 channels, albeit at lower activity levels-likely reflecting a smaller number of active channels. Overall, the NPo increased from 0.45 ± 0.23 to 1.69 ± 0.66 (Figure 1c).

Figure 1. Effect of cytochalasin D on TRPC6 channels overexpressed in CHO cells. (Shalygin A, et al., 2021)