Abstract
The PY and YXXphi motifs are canonical sorting signals involved in trafficking. Nedd4-2 and the mu(2)-subunit of the AP-2 complex target these motifs to facilitate internalization. Epithelial Na(+) channel (ENaC) subunits contain both motifs in their cytosolic COOH termini where they overlap ((S/T)PPPXYX(S/T)phi). Just preceding the PY and embedded within the YXXphi motifs are conserved serine/threonine. We test here whether these conserved Ser/Thr modulate ENaC activity by influencing the function of the internalization domains. We find that co-expression of dominant-negative dynamin (K44A) with ENaC increases channel activity. Conversely, co-expression of Nedd4-2 and epsin with ENaC decrease activity. Alanine substitution of the conserved Thr(628) preceding the PY motif in gamma-mENaC had no effect on basal activity. Channels with this mutation, however, responded to K44A and epsin but not Nedd4-2. Similarly, mutation of the proline repeat in the PY motif of gamma-mENaC disrupted only Nedd4-2 regulation having no effect on regulation by K44A and epsin. Alanine substitution of the conserved Thr within the YXX motif of gamma-mENaC (T635A) increased basal activity. Channels containing this mutation responded to Nedd4-2 but not K44A and epsin. Channels containing the T635(D/E) substitution in gamma-mENaC did not have increased basal activity and responded to Nedd4-2 but not K44A. The double mutant T628A,T635A did not respond to Nedd4-2 or K44A. Mutation of Thr(628) and Thr(635) also disrupted ENaC precipitation with the mu(2)-subunit of the AP-2 complex. Moreover, the YXXphi motif, independent of the PY motif, was sufficient to target degradation with T635A disrupting this effect. These results demonstrate that the overlapping PY and YXXphi motifs in ENaC are, in some instances, capable of independent function and that the Ser/Thr just preceding and within these domains impact this function.
Highlights
Function mutations result in inappropriate renal salt wasting in humans associated with electrolyte imbalance and decreased blood pressure (4 – 6)
The 2-subunit of the plasma membrane clathrin-associated adapter complex AP-2, and the analogous 1-subunit of the AP-1 complex associated with clathrin in the trans-Golgi network and endosome interact with proteins via YXX motifs [17,18,19,20]
Epithelial Na؉ channel (ENaC) Activity Is Differentially Modulated by Thr613 and Thr620, and Thr628 and Thr635 in - and ␥-mENaC, Respectively—Fig. 1A shows an alignment of the area including the overlapping PY and putative YXX motifs for mENaC subunits, as well as similar motifs conserved in the cytosolic COOH termini of Nav1, Kv11, barttin, and connexin 43 channel subunits
Summary
Materials—All chemicals were reagent grade and purchased from Sigma, BioMol, Fischer Scientific, and Tocris, unless noted otherwise. Truncation, deletion, and substitution mutagenesis of plasmids encoding ENaC and hSOS-␥A568-end-mENaC were performed using the QuikChange Site-directed Mutagenesis Kit (Stratagene). Mutants used in the current study included the truncation and deletion derivatives ␥637X and ␥⌬S613-R632, respectively, of hSOS-␥A568-end-mENaC. Because pSOS encodes a truncated hSOS incapable of membrane localization by itself, recombinant hSOSENaC fusion proteins only activate Ras signaling and promote yeast growth to complement when localized to the plasma membrane by information within the ENaC portion of the fusion protein. Current recordings were acquired with an Axopatch 200B (Axon Instruments, Union City, CA) interfaced via a Digidata 1322A (Axon Instruments) to a PC running the pClamp 9 suite of software (Axon Instruments) Both a family of test pulses (500 ms each) stepping by 20-mV increments form a holding potential of 40 to 100 mV to Ϫ120 mV and voltage ramps (500 ms) from 60 to Ϫ100 mV were used to measure ENaC activity. Unpaired data were compared using the appropriate t tests. p Ͻ 0.05 was considered significant
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