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

Abstract

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 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.

Molecular pathways mediating mechanical signaling in bone

“…Through this set of channels mechanical stimulus could induce membrane hyper and de/polarization or a complex multiphasic response. Indeed two stretchactivated membrane channels have been identified in bone cells (Duncan et al, 1996;Kizer et al, 1997). Cyclical strain has been shown to modulate the activity of certain channelschronically strained osteoblasts had significantly larger increases in whole cell conductance when subjected to additional mechanical strain than unstrained controls (Duncan and Hruska, 1994).…”

ATP masks stretch activation of epithelial sodium channels in A6 distal nephron cells

“…Nonetheless, the mechanosensitivity of ENaC remains a matter of controversy despite a variety of studies attempting to address this issue. Examples of studies in which ENaC appears to be stretch sensitive include the observations that the purified renal ENaC in planar lipid bilayer membranes can be activated by hydrostatic pressure (1,4,8) and that expression of ␣-ENaC in mammalian cells results in stretch-activated channel activity (10). Recent studies have shown that osmotic stretch regulates rat ␣-, ␤-, and ␥-ENaC expressed in Xenopus laevis oocytes (9).…”

Steroids and Exogenous γ-ENaC Subunit Modulate Cation Channels Formed by α-ENaC in Human B Lymphocytes

“…Human B Lymphocytes Express the Messages for the ␣ and ␤ Subunits of ENaC-Previous studies have demonstrated that expression of exogenous ␣-ENaC results in a stretch-activated cation channel (9). Nonetheless, we hypothesized that ␣-ENaC may be involved in forming the stretch-activated cation channel observed in human B lymphocytes.…”

“…In heterologous expression systems, maximal expression of the hENaC channel requires co-expression of all three subunits (7,8), and in oocytes, expression of ␣-ENaC cRNA alone produces little expression of any amiloride-sensitive currents. However, in other cell types, expression of the exogenous ␣-ENaC subunit alone can form a stretch-activated nonselective cation channel (9), and a similar nonselective cation channel has also been described in native lung epithelial alveolar type II cells (10 -12). This lung cation channel appears to be formed from ␣-ENaC alone and is equally permeable to Na ϩ and K ϩ , is sensitive to steroid hormones, and has a higher unit conductance (21 pS) than that of ␣␤␥-ENaC (4 -5 pS) (13,14).…”