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-activated, nonselective cation channels (SA-CAT) and increasing matrix protein production. SA-CAT channels are thought to be responsible for mechano-transduction 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 alpha-subunit of the epithelial sodium channel (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 indicated that the osteoblast clone's sequence was identical to that originally cloned from rat colon. The alpha-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 alpha-rENaC. Reconstitution of alpha-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.
Kizer
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Renal Division, Barnes-Jewish Hospital, Washington University Medical Center, St. Louis, MO 63110, USA.
Guo
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XL
Hruska
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K
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001853:Bone Marrow; D002118:Calcium / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D010006:Osteoblasts / Q000378:metabolism / Q000502:physiology*; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D015183:Restriction Mapping; D015222:Sodium Channels / Q000235:genetics* / Q000502:physiology*; D013314:Stress, Mechanical
D002412:Cations; D012333:RNA, Messenger; D015222:Sodium Channels; D000584:Amiloride; D002118:Calcium