Intersubunit conformational changes mediate epithelial sodium channel gating.

The epithelial Na(+) channel (ENaC) functions as a pathway for Na(+) absorption in the kidney and lung, where it is crucial for Na(+) homeostasis and blood pressure regulation. However, the basic mechanisms that control ENaC gating are poorly understood. Here we define a role in gating for residues forming interfaces between the extracellular domains of the three ENaC subunits. Using cysteine substitution combined with chemical cross-linking, we determined that residues located at equivalent positions in the three subunits (αK477, ßE446, and γE455) form interfaces with residues in adjacent subunits (ßV85, γV87, and αL120, respectively). Cross-linking of these residues altered ENaC activity in a length-dependent manner; long cross-linkers increased ENaC current by increasing its open probability, whereas short cross-linkers reduced ENaC open probability. Cross-linking also disrupted ENaC gating responses to extracellular pH and Na(+), signals which modulate ENaC activity during shifts in volume status. Introduction of charged side chains at the interfacing residues altered ENaC activity in a charge-dependent manner. Current increased when like charges were present at both interfacing residues, whereas opposing charges reduced current. Together, these data indicate that conformational changes at intersubunit interfaces participate in ENaC transitions between the open and closed states; movements that increase intersubunit distance favor the open state, whereas the closed state is favored when the distance is reduced. This provides a mechanism to modulate ENaC gating in response to changing extracellular conditions that threaten Na(+) homeostasis.

Neural Basis of Action Understanding: Evidence from Sign Language Aphasia.

BACKGROUND: The neural basis of action understanding is a hotly debated issue. The mirror neuron account holds that motor simulation in fronto-parietal circuits is critical to action understanding including speech comprehension, while others emphasize the ventral stream in the temporal lobe. Evidence from speech strongly supports the ventral stream account, but on the other hand, evidence from manual gesture comprehension (e.g., in limb apraxia) has led to contradictory findings. AIMS: Here we present a lesion analysis of sign language comprehension. Sign language is an excellent model for studying mirror system function in that it bridges the gap between the visual-manual system in which mirror neurons are best characterized and language systems which have represented a theoretical target of mirror neuron research. METHODS & PROCEDURES: Twenty-one life long deaf signers with focal cortical lesions performed two tasks: one involving the comprehension of individual signs and the other involving comprehension of signed sentences (commands). Participants' lesions, as indicated on MRI or CT scans, were mapped onto a template brain to explore the relationship between lesion location and sign comprehension measures. OUTCOMES & RESULTS: Single sign comprehension was not significantly affected by left hemisphere damage. Sentence sign comprehension impairments were associated with left temporal-parietal damage. We found that damage to mirror system related regions in the left frontal lobe were not associated with deficits on either of these comprehension tasks. CONCLUSIONS: We conclude that the mirror system is not critically involved in action understanding.

Ubiquitin-specific peptidase 8 (USP8) regulates endosomal trafficking of the epithelial Na+ channel.

Ubiquitination plays a key role in trafficking of the epithelial Na(+) channel (ENaC). Previous work indicated that ubiquitination enhances ENaC endocytosis and sorting to lysosomes for degradation. Moreover, a defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. In this work, we identified a role for USP8 in the control of ENaC ubiquitination and trafficking. USP8 increased ENaC current in Xenopus oocytes and collecting duct epithelia and enhanced ENaC abundance at the cell surface in HEK 293 cells. This resulted from altered endocytic sorting; USP8 abolished ENaC degradation in the endocytic pathway, but it had no effect on ENaC endocytosis. USP8 interacted with ENaC, as detected by co-immunoprecipitation, and it deubiquitinated ENaC. Consistent with a functional role for deubiquitination, mutation of the cytoplasmic lysines of ENaC reduced the effect of USP8 on ENaC cell surface abundance. In contrast to USP8, USP2-45 increased ENaC surface abundance by reducing endocytosis but not degradation. Thus, USP8 and USP2-45 selectively modulate ENaC trafficking at different steps in the endocytic pathway. Together with previous work, the data indicate that the ubiquitination state of ENaC is critical for the regulation of epithelial Na(+) absorption.