7 3 a r t I C l e SThe level of expression of voltage-gated calcium channels at the plasma membrane is a key regulator of calcium homeostasis in excitable cells, and of downstream effects such as calcium-dependent transcription 1,2 . Members of the high voltage-activated (HVA) calcium channel family are heteromultimeric protein complexes that contain a pore-forming α 1 subunit that defines the channel subtype, plus ancillary α 2 -δ and β subunits that not only alter the function of the α 1 subunit but also regulate the trafficking of the channel complex to the plasma membrane 3-8 . The mammalian genome encodes four different types of Cavβ subunit that have distinct spatial and temporal expression patterns [4][5][6] . With the exception of Cavβ 2a , these subunits are cytoplasmic proteins that physically bind to a region in the α 1 subunit domain I-II linker that is highly conserved among all HVA calcium channels and is termed the alpha interaction domain (AID) 7 . Crystal structure data show that the Cavβ subunit contains interacting SH3 and guanylate kinase domains, with the latter participating in high-affinity binding to the AID region [8][9][10] . The physiological consequences of this interaction are underscored by gene knockout studies showing that deletion of the Cavβ 1a or Cavβ 2a subunits causes embryonic lethality 11,12 and by findings that a premature stop mutation in Cavβ 4 causes an epileptic phenotype in mice 13 .It has been suggested that the Cavβ subunit masks an endoplasmic reticulum retention signal on the Cav2.1 α 1 subunit 14 , thereby leading to increased cell surface expression of P/Q-type channels. However, no specific endoplasmic reticulum retention motif in the AID and surrounding regions of the α 1 subunit has been identified, and removing the AID motif in the I-II linker of Cav2.1 does not increase current amplitude in the absence of Cavβ (ref. 15). Moreover, it is not clear whether different HVA calcium channel isoforms share common retention motifs. Here we show that Cav1.2 (L-type) calcium channels contain an endoplasmic reticulum retention motif in the proximal C-terminal region, and we provide evidence that the Cavβ subunit has a role in regulating proteasomal degradation of these channels. Our data show that the Cavβ subunit acts as a molecular switch that prevents the ubiquitination of the channels and their targeting to the ERAD complex and thereby regulates their expression at the plasma membrane. RESULTS Cavb regulates endoplasmic reticulum retention of Cav1.2We first performed an ELISA assay involving a Cav1.2 channel construct tagged with an extracellular hemagglutinin (HA) epitope (Fig. 1a). We compared immunoluminescence between permeabilized and nonpermeabilized cells, which allowed us to quantify the relative proportion of Cav1.2 channels that were inserted into the plasma membrane. Coexpression with the Cavβ 1b or Cavβ 2a subunit mediated a significant increase in the fraction of Cav1.2 channels at the cell surface (Fig. 1a and data not shown). This was confirmed by HA...

T-type calcium channels are essential contributors to the transmission of nociceptive signals in the primary afferent pain pathway. Here, we show that T-type calcium channels are ubiquitinated by WWP1, a plasma-membrane-associated ubiquitin ligase that binds to the intracellular domain III-IV linker region of the Cav3.2 T-type channel and modifies specific lysine residues in this region. A proteomic screen identified the deubiquitinating enzyme USP5 as a Cav3.2 III-IV linker interacting partner. Knockdown of USP5 via shRNA increases Cav3.2 ubiquitination, decreases Cav3.2 protein levels, and reduces Cav3.2 whole-cell currents. In vivo knockdown of USP5 or uncoupling USP5 from native Cav3.2 channels via intrathecal delivery of Tat peptides mediates analgesia in both inflammatory and neuropathic mouse models of mechanical hypersensitivity. Altogether, our experiments reveal a cell signaling pathway that regulates T-type channel activity and their role in nociceptive signaling.

Many epithelia, including the superficial epithelia of the airways, are thought to secrete “volume sensors,” which regulate the volume of the mucosal lining fluid. The epithelial Na + channel (ENaC) is often the rate limiting factor in fluid absorption, and must be cleaved by extracellular and/or intracellular proteases before it can conduct Na + and absorb excess mucosal liquid, a process that can be blocked by proteases inhibitors. In the airways, airway surface liquid dilution or removal activates ENaC. Therefore, we hypothesized that endogenous proteases are membrane-anchored, whereas endogenous proteolysis inhibitors are soluble and can function as airway surface liquid volume sensors to inhibit ENaC activity. Using a proteomic approach, we identified short palate, lung, and nasal epithelial clone (SPLUNC)1 as a candidate volume sensor. Recombinant SPLUNC1 inhibited ENaC activity in both human bronchial epithelial cultures and Xenopus oocytes. Knockdown of SPLUNC1 by shRNA resulted in a failure of bronchial epithelial cultures to regulate ENaC activity and airway surface liquid volume, which was restored by adding recombinant SPLUNC1 to the airway surface liquid. Despite being able to inhibit ENaC, recombinant SPLUNC1 had little effect on extracellular serine protease activity. However, SPLUNC1 specifically bound to ENaC, preventing its cleavage and activation by serine proteases. SPLUNC1 is highly expressed in the airways, as well as in colon and kidney. Thus, we propose that SPLUNC1 is secreted onto mucosal surfaces as a soluble volume sensor whose concentration and dilution can regulate ENaC activity and mucosal volumes, including that of airway surface liquid.

Epithelial sodium channels (ENaCs) perform diverse physiological roles by mediating Na+ absorption across epithelial surfaces throughout the body. Excessive Na+ absorption in kidney and colon elevates blood pressure and in the airways disrupts mucociliary clearance. Potential therapies for disorders of Na+ absorption require better understanding of ENaC regulation. Recent work has established partial and selective proteolysis of ENaCs as an important means of channel activation. In particular, channel-activating transmembrane serine proteases (CAPs) and cognate inhibitors may be important in tissue-specific regulation of ENaCs. Although CAP2 (TMPRSS4) requires catalytic activity to activate ENaCs, there is not yet evidence of ENaC fragments produced by this serine protease and/or identification of the site(s) where CAP2 cleaves ENaCs. Here, we report that CAP2 cleaves at multiple sites in all three ENaC subunits, including cleavage at a conserved basic residue located in the vicinity of the degenerin site (α-K561, β-R503, and γ-R515). Sites in α-ENaC at K149/R164/K169/R177 and furin-consensus sites in α-ENaC (R205/R231) and γ-ENaC (R138) are responsible for ENaC fragments observed in oocytes coexpressing CAP2. However, the only one of these demonstrated cleavage events that is relevant for the channel activation by CAP2 takes place in γ-ENaC at position R138, the previously identified furin-consensus cleavage site. Replacement of arginine by alanine or glutamine (α,β,γR138A/Q) completely abolished both the Na+ current (INa) and a 75-kD γ-ENaC fragment at the cell surface stimulated by CAP2. Replacement of γ-ENaC R138 with a conserved basic residue, lysine, preserved both the CAP2-induced INa and the 75-kD γ-ENaC fragment. These data strongly support a model where CAP2 activates ENaCs by cleaving at R138 in γ-ENaC.

The epithelial sodium channel (ENaC) is preferentially assembled into heteromeric ␣␤␥ complexes. The ␣ and ␥ (not ␤) subunits undergo proteolytic cleavage by endogenous furinlike activity correlating with increased ENaC function. We identified full-length subunits and their fragments at the cell surface, as well as in the intracellular pool, for all homo-and heteromeric combinations (␣, ␤, ␥, ␣␤, ␣␥, ␤␥, and ␣␤␥). We assayed corresponding channel function as amiloride-sensitive sodium transport (I Na ). We varied furin-mediated proteolysis by mutating the P1 site in ␣ and/or ␥ subunit furin consensus cleavage sites (␣ mut and ␥ mut ). Our findings were as follows. (i) The ␤ subunit alone is not transported to the cell surface nor cleaved upon assembly with the ␣ and/or ␥ subunits. (ii) The ␣ subunit alone (or in combination with ␤ and/or ␥) is efficiently transported to the cell surface; a surface-expressed 65-kDa ␣ ENaC fragment is undetected in ␣ mut ␤␥, and I Na is decreased by 60%. (iii) The ␥ subunit alone does not appear at the cell surface; ␥ co-expressed with ␣ reaches the surface but is not detectably cleaved; and ␥ in ␣␤␥ complexes appears mainly as a 76-kDa species in the surface pool. Although basal I Na of ␣␤␥ mut was similar to ␣␤␥, ␥ mut was not detectably cleaved at the cell surface. Thus, furin-mediated cleavage is not essential for participation of ␣ and ␥ in ␣␤␥ heteromers. Basal I Na is reduced by preventing furin-mediated cleavage of the ␣, but not ␥, subunits. Residual current in the absence of furin-mediated proteolysis may be due to non-furin endogenous proteases.The highly sodium-selective epithelial sodium channel (ENaC) 2 is composed of three homologous subunits (␣, ␤, and ␥) and is expressed in several ion-transporting epithelia, including the kidney, colon, and lung (1). The membrane topology of each subunit was predicted to consist of two transmembrane domains with short cytoplasmic amino and carboxyl termini and joined by a large extracellular loop. This prediction is strongly supported by recent crystallization of ENaC relative ASIC1. The ASIC1 structure (2) also makes it clear that ENaC is likely a heterotrimer (␣ 1 ␤ 1 ␥ 1 ), in contrast to the heterotetrameric, octameric, or nonameric stoichiometry (␣␤␣␥) proposed previously (3-5). In the Xenopus oocyte expression system, it has been possible to quantitate the number of channel complexes expressed at the cell surface and to measure in the same oocyte the amiloride-sensitive current generated by the expression of ENaC at the membrane (6, 7). This assay demonstrated the preferential assembly of ␣␤␥ channels. Maximal ENaC activity measured by the amiloride-sensitive inward current (I Na ) is observed only when the ␣, ␤, or ␥ subunits are co-injected in the Xenopus oocyte expression system. There is an excellent correlation between I Na and the number of channel complexes expressed at the cell surface (7). When the ␣ subunit is injected alone (presumably ␣3 trimers), only 1 or 2% of maximal activity is observed. When the ␣/␤ or ␣...