Steroids and exogenous gamma-ENaC subunit modulate cation channels formed by alpha-ENaC in human B lymphocytes.
Previous studies using whole-cell recording methods suggest that human B lymphocytes express an amiloride-sensitive, sodium-permeable channel. The present studies aim to determine whether this channel has biophysical properties and a molecular structure related to the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC). Reverse transcriptase polymerase chain reaction and Northern blots showed that human B lymphocytes express messages for both alpha- and beta- but not gamma-ENaC. Western blots showed that both alpha- and beta- but not gamma-ENaC proteins are expressed and strongly reduced by antisense oligonucleotides. Patch clamp experiments demonstrated that lymphocyte sodium channels are not active in cell-attached patches. However, membrane stretch can activate a 21-pS nonselective cation channel. The frequency of observance of this channel was significantly reduced by antisense oligonucleotide against alpha-ENaC but not by antisense oligonucleotide against beta-ENaC, indicating that only the alpha subunit of ENaC is necessary to form stretch-activated cation channels. Aldosterone (1.5 microm) reduced the frequency of observance of 21-pS alpha-ENaC channels and simultaneously induced the appearance of spontaneously active 10-pS channels. Antisense oligonucleotide experiments showed that this 10-pS channel is formed from alpha- and beta-ENaC. After expression of exogenous gamma-ENaC, aldosterone again reduced the frequency of observance of the 21-pS alpha-ENaC channel but induced the appearance of a 5-pS channel, presumably a alphabetagamma-ENaC channel. In the absence of aldosterone, the alpha subunit forms an alpha-cryptic channel that is activated by stretch, and in the presence of aldosterone, beta and alpha subunits together form an active channel that is modulated by aldosterone.
Ma
He-Ping
HP
Center for Cell and Molecular Signaling and Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA. hma@physio.emory.edu
Al-Khalili
Otor
O
Ramosevac
Semra
S
Saxena
Sunil
S
Liang
You-You
YY
Warnock
David G
DG
Eaton
Douglas C
DC
D000450:Aldosterone / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D001402:B-Lymphocytes / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D004553:Electric Conductivity; D053503:Epithelial Sodium Channels; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008465:Mechanoreceptors / Q000502:physiology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015222:Sodium Channels / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics / Q000494:pharmacology* / Q000502:physiology*; D013256:Steroids / Q000494:pharmacology*; D014162:Transfection
D053503:Epithelial Sodium Channels; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D015222:Sodium Channels; D013256:Steroids; D000450:Aldosterone; D000584:Amiloride