ENaCγ亚基对Na+的稳态至关重要,K+,和体液。在短抑制道之前和之后的双γ亚基裂解允许该道的解离,增加信道开放概率(PO),在体外。在furin识别序列(143RKRR146,在小鼠γ亚基中)发生在管道附近的裂解。弗林蛋白酶介导的裂解的丧失阻止了通过远端位点的蛋白水解对通道的体外激活。我们假设在129/Sv小鼠中143RKRR146突变为143QQQQ146(γQ4)会降低ENaCPO,削弱灌注收集管中Na+(JNa)和K+(JK)的流动刺激通量,降低对结肠阿米洛利敏感的短路电流(ISC),并损害Na+,K+,和体液稳态。γQ4/Q4小鼠肾裂解物的免疫印迹证实了与弗林蛋白酶切割的蛋白水解片段大小一致的条带的丢失。然而,γQ4/Q4雄性小鼠在低Na+饮食中没有表现出改变的ENaCPO或流动诱导的JNa,尽管流量诱导的JK略有下降。γQ4/Q4小鼠对结肠阿米洛利敏感的ISC没有改变。γQ4/Q4男性,但不是女性,当受到低Na饮食的挑战时,表现出轻度损害的液体体积保守性。血液Na+和K+在常规上没有变化,低Na+,或高K+饮食。这些发现表明,应将γ亚基裂解的生化证据用于分离以评估ENaC活性。Further,独立于γ亚基裂解的因子调节通道PO和ENaC对Na+的影响,K+,129/Sv小鼠的液体体积稳态,在体内。
The epithelial Na+ channel (
ENaC) γ subunit is essential for homeostasis of Na+, K+, and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146, in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (γQ4) in 129/Sv mice would reduce
ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of γQ4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered
ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate
ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel PO and the influence of
ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na+ or high K+ diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC\'s γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na+ or high K+ diets.