Expression and regulation of normal and polymorphic epithelial sodium channel by human lymphocytes

Gene expression, protein expression, and function of amiloride-sensitive so dium channels were examined in human lymphocytes from normal individuals an d individuals with Liddle's disease. Using reverse transcriptase polymerase chain reactions, expression of all three cloned epithelial sodium channel (ENaC) subunits was detected in lymphocytes. Polyclonal antibodies to bovin e alpha -ENaC bound to the plasma membrane of normal and Liddle's lymphocyt es. A quantitative analysis of fluorescence-tagged ENaC antibodies indicate d a 2.5-fold greater surface binding of the antibodies to Liddle's lymphocy tes compared with normal lymphocytes. The relative binding intensity increa sed significantly (25%; p < 0.001) for both normal and Liddle's cells after treatment with 40 <mu>M 8-CPT-cAMP. Amiloride-sensitive whole cell current s were recorded under basal and cAMP-treated conditions for both cell types . Liddle's cells had a 4.5-fold larger inward sodium conductance compared w ith normal cells. A specific 25% increase in the inward sodium current was observed in normal cells in response to cAMP treatment. Outside-out patches from both cell types under both treatment conditions revealed no obvious d ifferences in the single channel conductance. The P-open was 4.2 +/- 3.9% f or patches from non-Liddle's cells, and 27.7 +/- 5.4% in patches from Liddi e's lymphocytes. Biochemical purification of a protein complex, using the s ame antibodies used for the immunohistochemistry, yielded a functional sodi um channel complex that was inhibited by amiloride when reconstituted into lipid vesicles and incorporated into planar lipid bilayers, These four inde pendent methodologies yielded findings consistent with the hypotheses that human lymphocytes express functional, regulatable ENaC and that the mutatio n responsible for Liddle's disease induces excessive channel expression.