Reconstitution of stretch-activated cation channels by expression of the α-subunit of the epithelial sodium channel cloned from osteoblasts

Osteoblasts respond to repetitive strain by activating stretch- activated, nonselective cation channels (SA-CAT) and increasing matrix protein production. SA-CAT channels are thought to be responsible for mechanotransduction in osteoblasts, although the molecular identity of the SA-CAT channel has previously been unknown. We have demonstrated that both the UMR-106 osteoblast-like cell line and human osteoblasts in primary culture express the α-subunit of the epithelial sodium channel (α-ENaC). The ENaC gene product is closely related to a class of proteins that confer touch sensitivity to Caenorhabditis elegans and are referred to as degenerins. A cDNA clone was obtained of the entire coding region of rat α- ENaC (α-rENaC). Sequence analysis indicated that the osteoblast clone's sequence was identical to that originally cloned from rat colon. The α- rENaC cDNA was cloned into an expression plasmid and transfected into LM(TK^- ) cells, a null cell for SA-CAT activity. Stable transfectants expressed mRNA and the expected 74-kDa protein corresponding to α-rENaC. Reconstitution of α-rENaC resulted in the expression of a 24.2 ± 1.0 psec SA-CAT channel (P(Na):P(K) = 1.1 ± 0.1). The channel is calcium permeable (P(Na):P(Ca) = 1.4 ± 0.1) and highly selective for cations over anions (P(Na):P(Cl) >> 20). The channel is only active after negative pressure is applied to cell attached patches, cell swelling, or patch excision. These results represent the first heterologous expression of an SA-CAT channel in a mammalian cell system and provide evidence that the ENaC/degenerin family of proteins are capable of mediating both transepithelial sodium transport and are involved in signal transduction by mechano-sensitive cells such as osteoblasts.