The Spectrin Cytoskeleton Influences the Surface Expression and Activation of Human Transient Receptor Potential Channel 4 Channels

Abstract

Despite over a decade of research, only recently have the mechanisms governing transient receptor potential channel (TRPC) channel function begun to emerge, with an essential role for accessory proteins in this process. We previously identified a tyrosine phosphorylation event as critical in the plasma membrane translocation and activation of hTRPC4 channels following epidermal growth factor (EGF) receptor activation. To further characterize the signaling events underlying this process, a yeast-two hybrid screen was performed on the C terminus of hTRPC4. The intracellular C-terminal region from proline 686 to leucine 977 was used to screen a human brain cDNA library. Two members of the spectrin family, alphaII- and betaV-spectrin, were identified as binding partners. The interaction of hTRPC4 with alphaII-spectrin and betaV-spectrin was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation experiments. Deletion analysis identified amino acids 730-758 of hTRPC4 as critical for the interaction with this region located within a coiled-coil domain, juxtaposing the Ca(2+)/calmodulin- and IP(3)R-binding region (CIRB domain). This region is deleted in the proposed deltahTRPC4 splice variant form, which failed to undergo both EGF-induced membrane insertion and activation, providing a genetic mechanism for regulating channel activity. We also demonstrate that the exocytotic insertion and activation of hTRPC4 following EGF application is accompanied by dissociation from alphaII-spectrin. Furthermore, depletion of alphaII-spectrin by small interference RNA reduces the basal surface expression of alphahTRPC4 and prevents the enhanced membrane insertion in response to EGF application. Importantly, depletion of alphaII-spectrin did not affect the expression of the delta variant. Taken together, these results demonstrate that a direct interaction between hTRPC4 and the spectrin cytoskeleton is involved in the regulation of hTRPC4 surface expression and activation.