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. 2016 Nov 28;292(1):375–385. doi: 10.1074/jbc.M116.753616

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FIGURE 6. — AnkG alters ENaC activity by increasing the rate of surface delivery. A, representative I_sc trace of mCCD cell current recovery after 5 μm forskolin stimulation and block by 25 μm phenamil. Phenamil irreversibly blocks all channels at the surface, and once the phenamil has been removed, the amiloride-sensitive current that appears over time is the result of channels being inserted from the biosynthetic and non-cAMP-regulated pathway. Pretreatment with forskolin inserts channels from the recycling pool into the surface, eliminating them as possible contributions to the recovery current. B, current recovery plotted as a percentage of preinhibited values over time. The rate and extent of recovery in AnkG OE cells is greater than control or AnkG KD mCCDs. C, percentage recovery summary demonstrated that after 30 min, significantly more channels were delivered to the surface with AnkG OE (24 ± 2.5%, n = 9; p < 0.01) and significantly fewer channels with AnkG KD (8 ± 2.8%, n = 8, p value<0.05) relative to control (15 ± 1.8%, n = 20). D, representative I_sc trace of FRTs expressing CR ENaC in combination with Liddle's mutation with or without AnkG OE. Following trypsin activation and washout, electrically silent channels were being delivered to the plasma membrane during the current run down time course. After 30 min, the cells were restimulated with trypsin. The increase from the base current is indicative of the number of channels delivered to the surface during the 30-min time period. E, summarized I_sc increase for the second trypsin pulse following the 30-min interval from n > 4 experiments like those in Figs. 4B and 6D, respectively. AnkG overexpression delivered 75% ± 16% more ENaC in 30 min than control cells (n = 4, p < 0.001) without the Liddle's mutation, and this increase was absent with the β-ENaC Liddle's mutant. F, absolute I_sc values following the second trypsin pulse. AnkG OE did not significantly change the number of channels that are being delivered from the biosynthetic pathway because no significant increase in delivery of silent ENaC was observed with AnkG OE with the Liddle's mutation, as was the case in non-Liddle's ENaC.