Potent functional uncoupling between STIM1 and Orai1 by dimeric 2-aminodiphenyl borinate analogs.

The coupling of ER Ca(2+)-sensing STIM proteins and PM Orai Ca(2+) entry channels generates "store-operated" Ca(2+) signals crucial in controlling responses in many cell types. The dimeric derivative of 2-aminoethoxydiphenyl borinate (2-APB), DPB162-AE, blocks functional coupling between STIM1 and Orai1 with an IC50 (200 nM) 100-fold lower than 2-APB. Unlike 2-APB, DPB162-AE does not affect L-type or TRPC channels or Ca(2+) pumps at maximal STIM1-Orai1 blocking levels. DPB162-AE blocks STIM1-induced Orai1 or Orai2, but does not block Orai3 or STIM2-mediated effects. We narrowed the DPB162-AE site of action to the STIM-Orai activating region (SOAR) of STIM1. DPB162-AE does not prevent the SOAR-Orai1 interaction but potently blocks SOAR-mediated Orai1 channel activation, yet its action is not as an Orai1 channel pore blocker. Using the SOAR-F394H mutant which prevents both physical and functional coupling to Orai1, we reveal DPB162-AE rapidly restores SOAR-Orai binding but only slowly restores Orai1 channel-mediated Ca(2+) entry. With the same SOAR mutant, 2-APB induces rapid physical and functional coupling to Orai1, but channel activation is transient. We infer that the actions of both 2-APB and DPB162-AE are directed toward the STIM1-Orai1 coupling interface. Compared to 2-APB, DPB162-AE is a much more potent and specific STIM1/Orai1 functional uncoupler. DPB162-AE provides an important pharmacological tool and a useful mechanistic probe for the function and coupling between STIM1 and Orai1 channels.

Initial steps of inactivation at the K+ channel selectivity filter.

K(+) efflux through K(+) channels can be controlled by C-type inactivation, which is thought to arise from a conformational change near the channel's selectivity filter. Inactivation is modulated by ion binding near the selectivity filter; however, the molecular forces that initiate inactivation remain unclear. We probe these driving forces by electrophysiology and molecular simulation of MthK, a prototypical K(+) channel. Either Mg(2+) or Ca(2+) can reduce K(+) efflux through MthK channels. However, Ca(2+), but not Mg(2+), can enhance entry to the inactivated state. Molecular simulations illustrate that, in the MthK pore, Ca(2+) ions can partially dehydrate, enabling selective accessibility of Ca(2+) to a site at the entry to the selectivity filter. Ca(2+) binding at the site interacts with K(+) ions in the selectivity filter, facilitating a conformational change within the filter and subsequent inactivation. These results support an ionic mechanism that precedes changes in channel conformation to initiate inactivation.

Distinct Orai-coupling domains in STIM1 and STIM2 define the Orai-activating site.

STIM1 and STIM2 are widely expressed endoplasmic reticulum (ER) Ca(2+) sensor proteins able to translocate within the ER membrane to physically couple with and gate plasma membrane Orai Ca(2+) channels. Although they are structurally similar, we reveal critical differences in the function of the short STIM-Orai-activating regions (SOAR) of STIM1 and STIM2. We narrow these differences in Orai1 gating to a strategically exposed phenylalanine residue (Phe-394) in SOAR1, which in SOAR2 is substituted by a leucine residue. Remarkably, in full-length STIM1, replacement of Phe-394 with the dimensionally similar but polar histidine head group prevents both Orai1 binding and gating, creating an Orai1 non-agonist. Thus, this residue is critical in tuning the efficacy of Orai activation. While STIM1 is a full Orai1-agonist, leucine-replacement of this crucial residue in STIM2 endows it with partial agonist properties, which may be critical for limiting Orai1 activation stemming from its enhanced sensitivity to store-depletion.

The calcium store sensor, STIM1, reciprocally controls Orai and CaV1.2 channels.

Calcium signals, pivotal in controlling cell function, can be generated by calcium entry channels activated by plasma membrane depolarization or depletion of internal calcium stores. We reveal a regulatory link between these two channel subtypes mediated by the ubiquitous calcium-sensing STIM proteins. STIM1 activation by store depletion or mutational modification strongly suppresses voltage-operated calcium (Ca(V)1.2) channels while activating store-operated Orai channels. Both actions are mediated by the short STIM-Orai activating region (SOAR) of STIM1. STIM1 interacts with Ca(V)1.2 channels and localizes within discrete endoplasmic reticulum/plasma membrane junctions containing both Ca(V)1.2 and Orai1 channels. Hence, STIM1 interacts with and reciprocally controls two major calcium channels hitherto thought to operate independently. Such coordinated control of the widely expressed Ca(V)1.2 and Orai channels has major implications for Ca(2+) signal generation in excitable and nonexcitable cells.

Intrinsic voltage dependence of the epithelial Na+ channel is masked by a conserved transmembrane domain tryptophan.

Tryptophan residues critical to function are frequently located at the lipid-water interface of transmembrane domains. All members of the epithelial Na+ channel (ENaC)/Degenerin (Deg) channel superfamily contain an absolutely conserved Trp at the base of their first transmembrane domain. Here, we test the importance of this conserved Trp to ENaC/Deg function. Targeted substitution of this Trp in mouse ENaC and rat ASIC subunits decrease channel activity. Differential substitution with distinct amino acids in alpha-mENaC shows that it is loss of this critical Trp rather than introduction of residues having novel properties that changes channel activity. Surprisingly, Trp substitution unmasks voltage sensitivity. Mutant ENaC has increased steady-state activity at hyperpolarizing compared with depolarizing potentials associated with transient activation and deactivation times, respectively. The times of activation and deactivation change 1 ms/mV in a linear manner with rising and decreasing slopes, respectively. Increases in macroscopic currents at hyperpolarizing potentials results from a voltage-dependent increase in open probability. Voltage sensitivity is not influenced by divalent cations; however, it is Na+-dependent with a 63-mV decrease in voltage required to reach half-maximal activity per log increase in [Na+]. Mutant channels are particularly sensitive to intracellular [Na+] for removing this sodium abolishes voltage dependence. We conclude that the conserved Trp at the base of TM1 in ENaC/Deg channels protects against voltage by masking an inhibitory allosteric or pore block mechanism, which decreases activity in response to intracellular Na+.

STIM protein coupling in the activation of Orai channels.

STIM proteins are sensors of endoplasmic reticulum (ER) luminal Ca(2+) changes and rapidly translocate into near plasma membrane (PM) junctions to activate Ca(2+) entry through the Orai family of highly Ca(2+)-selective "store-operated" channels (SOCs). Dissecting the STIM-Orai coupling process is restricted by the abstruse nature of the ER-PM junctional domain. To overcome this problem, we studied coupling by using STIM chimera and cytoplasmic C-terminal domains of STIM1 and STIM2 (S1ct and S2ct) and identifying a fundamental action of the powerful SOC modifier, 2-aminoethoxydiphenyl borate (2-APB), the mechanism of which has eluded recent scrutiny. We reveal that 2-APB induces profound, rapid, and direct interactions between S1ct or S2ct and Orai1, effecting full Ca(2+) release-activated Ca(2+) (CRAC) current activation. The short 235-505 S1ct coiled-coil region was sufficient for functional Orai1 coupling. YFP-tagged S1ct or S2ct fragments cleared from the cytosol seconds after 2-APB addition, binding avidly to Orai1-CFP with a rapid increase in FRET and transiently increasing CRAC current 200-fold above basal levels. Functional S1ct-Orai1 coupling occurred in STIM1/STIM2(-/-) DT40 chicken B cells, indicating ct fragments operate independently of native STIM proteins. The 2-APB-induced S1ct-Orai1 and S2-ct-Orai1 complexes undergo rapid reorganization into discrete colocalized PM clusters, which remain stable for >100 s, well beyond CRAC activation and subsequent deactivation. In addition to defining 2-APB's action, the locked STIMct-Orai complex provides a potentially useful probe to structurally examine coupling.

Activation of mitogen-activated protein kinase (mitogen-activated protein kinase/extracellular signal-regulated kinase) cascade by aldosterone.

Aldosterone in some tissues increases expression of the mRNA encoding the small monomeric G protein Ki-RasA. Renal A6 epithelial cells were used to determine whether induction of Ki-ras leads to concomitant increases in the total as well as active levels of Ki-RasA and whether this then leads to subsequent activation of its effector mitogen-activated protein kinase (MAPK/extracellular signal-regulated kinase) cascade. The molecular basis and cellular consequences of this action were specifically investigated. We identified the intron 1-exon 1 region (rasI/E1) of the mouse Ki-ras gene as sufficient to reconstitute aldosterone responsiveness to a heterologous promotor. Aldosterone increased reporter gene activity containing rasI/E1 threefold. Aldosterone increased the absolute and GTP-bound levels of Ki-RasA by a similar extent, suggesting that activation resulted from mass action and not effects on GTP binding/hydrolysis rates. Aldosterone significantly increased Ki-RasA and MAPK activity as early as 15 min with activation peaking by 2 h and waning after 4 h. Inhibitors of transcription, translation, and a glucocorticoid receptor antagonist attenuated MAPK signaling. Similarly, rasI/E1-driven luciferase expression was sensitive to glucocorticoid receptor blockade. Overexpression of dominant-negative RasN17, addition of antisense Ki-rasA and inhibition of mitogen-activated protein kinase kinase also attenuated steroid-dependent increases in MAPK signaling. Thus, activation of MAPK by aldosterone is dependent, in part, on a genomic mechanism involving induction of Ki-ras transcription and subsequent activation of its downstream effectors. This genomic mechanism has a distinct time course from activation by traditional mitogens, such as serum, which affect the GTP-binding state and not absolute levels of Ras. The result of such a genomic mechanism is that peak activation of the MAPK cascade by adrenal corticosteroids is delayed but prolonged.

Identification of cytoplasmic domains within the epithelial Na+ channel reactive at the plasma membrane.

The activity of membrane proteins is controlled, in part, by protein-protein interactions localized to the plasma membrane. In the current study, domains within the epithelial Na(+) channel (ENaC) reactive at the plasma membrane were identified using a novel yeast one-hybrid screen. The cytosolic N terminus of alphaENaC and the cytosolic C termini of alpha-, beta-, and gammaENaC contained domains reactive at the plasma membrane. Fluorescent micrographs of epithelial cells overexpressing fusion proteins of enhanced green fluorescent protein and mENaC cytosolic domains were consistent with those in yeast. A novel membrane reactive domain within the cytosolic C terminus of gamma-mENaC was localized to the 17 amino acids between residues Thr(584)-Pro(600). Two overlapping internalization signals within the C terminus of gamma-mENaC, a WW-binding domain (PY motif) and a tyrosine-based endocytic signal, were additive with respect to decreasing complementation and expression levels of hybrid proteins. Decreases in expression levels of hybrid proteins containing the PY and endocytic motif were reversed with latrunculin A, an inhibitor of endosomal movement. Decreases in complementation and expression levels of hybrid proteins mediated by the combined PY and overlapping endocytic motif proceeded in the absence of established ubiquitination sites within ENaC. In addition, the endocytic motif was active in the absence of the PY motif, demonstrating that these two domains, while possibly interacting, also have discrete functions. The novel domains within the cytosolic N terminus of alphaENaC and the C termini of alpha-, beta-, and gammaENaC identified here are likely to be involved in protein-protein and/or protein-lipid interactions localized to the plasma membrane. We hypothesize that these newly identified domains play a role in modulating ENaC activity.