T1845 Enhanced Mucosal Expression of KCNN4 and Bk Channels Contribute to Aldosterone Induced K+ Secretion in Distal Colon

Abstract

Background: Mammalian colon plays important roles in body K+ homeostasis, as it both actively absorbs and secretes K+. Aldosterone (aldo) stimulates both active electroneutral K+ absorption and active electrogenic K+ secretion in rat distal colon. Increased H,K-ATPase expression has been shown as a mechanism for enhanced K+ absorption. However, although both intermediate conductance (KCNN4) and large conductance (BK) K+ channels are present on the mucosal membranes, a mechanism responsible for aldo increased K+ secretion is not known. Aim: Studies were initiated to identify whether KCNN4 and/or BK channels mediate the aldo enhanced K+ secretion in rat distal colon. Methods: Aldo animals were produced by feeding Na-free diet for 6-7 days. Mucosal to serosal (m-s) and serosal to mucosal (s-m) 86Rb (K+ surrogate) fluxes were measured in stripped colonic mucosa mounted under voltage clamp condition in presence of mucosal benzamil (50 μM; ENaC blocker). Western blot analyses were performed using anti-KCNN4-abc (developed in our laboratory) and BKα (Santa Cruz) antibodies. RTQ-PCR analyses were performed using standard technique. Results: Under basal condition, mucosal addition of VO4 (H,K-ATPase inhibitor) inhibited the net K+ absorption (0.42 ± 0.06 vs -0.04 ± 0.03 μEq/h.cm2; p < 0.001) in normal rat distal colon. Serosal addition of carbachol (CCH) induced net K secretion (-0.04 ± 0.03 vs 0.48 ± 0.06 μEq/h.cm2; p < 0.001). The CCH-induced net K+ secretion was inhibited by 60% and 40% by mucosal TRAM-34 (KCNN4 channel blocker) and iberiotoxin (IbTX; BK channel blocker), respectively. Either simultaneous presence of TRAM-34 and IbTX or Ba2+ (nonspecific K+ channel blocker) completely inhibited the CCH-induced K secretion. Aldo reversed the net K absorption in normal colon to net K+ secretion (0.42 ± 0.06 vs -1.34 ± 0.22 μEq/h.cm2; p < 0.001). Mucosal addition of TRAM34 and IbTX inhibited the aldo-induced net K+ secretion by 33% and 55%, respectively. Simultaneous presence of TRMA-34 and IbTX, and Ba2+ inhibited the aldo induced net K secretion by 84% and 80%, respectively. Western blot analyses indicated that aldo enhanced mucosal KCNN4 and BKα specific protein expression by 2.2 and 3.1 fold, respectively. RTQ-PCR analyses indicated that mucosal KCNN4 and BKα channel specific mRNA abundances were increased by 3.0 and 3.5 fold in aldo animals, respectively. Conclusions: 1) Both KCNN4 and BK channels mediate CCHand aldo-induced K+ secretion; 2) Enhanced expression of both mucosal KCNN4 and BK channels mediate the aldo induced K+ secretion; and 3) Aldo regulates both KCNN4 and BK channel expression at transcriptional level.