Immunopurification and functional reconstitution of a Na+ channel complex from rat lymphocytes

Academic Article

Abstract

  • Patch-clamp experiments have demonstrated an amiloride-sensitive Na+ conductance in human B lymphoid cells. We measured whole cell currents in rat lymphocytes and observed a similar Na+-specific inward conductance. The presence of 400 μM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate in the bath significantly increased the inward current, and this adenosine 3',5'-cyclic monophosphate activation was abolished by 2 μM amiloride. We immunopurified a protein complex from rat lymphocyte membranes using an anti-bovine kidney Na+ channel antibody. The complex consisted of five distinct polypeptides with apparent M(r) values of 110,000, 92,000, 59,000, 48,000, and 42,000. This putative channel complex was incorporated into planar lipid bilayers, where we observed single Na+ channel activity that was blocked by amiloride in a concentration-dependent manner. The addition of protein kinase A and ATP to the 'intracellular' solution elicited a twofold increase in channel activity. Reverse transcription-polymerase chain reaction analysis was used to determine if the rat lymphocytes express the message for the recently cloned Na+ channel of the rat colon (rENaC). Primers for the α-subunit of rENaC identified no message in the lymphocyte RNA, while primers for the β-subunit of the clone produced low levels of the expected product. Thus it appears that a rENaC-like β-subunit may be an essential component of the lymphocyte Na+ channel that was isolated. At the same time, this channel is different from those recently cloned in that it does not include an α-subunit homologous to that of rENaC.
  • Authors

    Digital Object Identifier (doi)

    Author List

  • Bradford AL; Ismailov II; Achard JM; Warnock DG; Bubien JK; Benos DJ
  • Volume

  • 269
  • Issue

  • 3 38-3