RECONSTITUTION OF STRETCH-ACTIVATED CATION CHANNELS BY EXPRESSION OF THE ALPHA-SUBUNIT OF THE EPITHELIAL SODIUM-CHANNEL CLONED FROM OSTEOBLASTS

Osteoblasts respond to repetitive strain by activating stretch-activat ed, nonselective cation channels (SA-CAT) and increasing matrix protei n production. SA-CAT channels are thought to be responsible for mechan otransduction in osteoblasts, although the molecular identity of the S A-CAT channel has previously been unknown. We have demonstrated that b oth the UMR-106 osteoblast-like cell line and human osteoblasts in pri mary culture express the alpha-subunit of the epithelial sodium channe l (alpha-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 alpha-ENaC (alpha-rENaC). Sequence analysis indi cated that the osteoblast clone's sequence was identical to that origi nally cloned from rat colon. The alpha-rENaC cDNA was cloned into an e xpression 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 alpha-rENaC. Reconstitution of alpha-r ENaC 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 much greater than 20) The channel is only active after negative press ure is applied to cell attached patches, cell swelling, or patch excis ion. These results represent the first heterologous expression of an S A-CAT channel in a mammalian cell system and provide evidence that the ENaC/degenerin family of proteins are capable of mediating both trans epithelial sodium transport and are involved in signal transduction by mechano sensitive cells such as osteoblasts.