Grp94 Protein Delivers γ-Aminobutyric Acid Type A (GABAA) Receptors to Hrd1 Protein-mediated Endoplasmic Reticulum-associated Degradation.

Proteostasis maintenance of γ-aminobutyric acid type A (GABAA) receptors dictates their function in controlling neuronal inhibition in mammalian central nervous systems. However, as a multisubunit, multispan, integral membrane protein, even wild type subunits of GABAA receptors fold and assemble inefficiently in the endoplasmic reticulum (ER). Unassembled and misfolded subunits undergo ER-associated degradation (ERAD), but this degradation process remains poorly understood for GABAA receptors. Here, using the α1 subunits of GABAA receptors as a model substrate, we demonstrated that Grp94, a metazoan-specific Hsp90 in the ER lumen, uses its middle domain to interact with the α1 subunits and positively regulates their ERAD. OS-9, an ER-resident lectin, acts downstream of Grp94 to further recognize misfolded α1 subunits in a glycan-dependent manner. This delivers misfolded α1 subunits to the Hrd1-mediated ubiquitination and the valosin-containing protein-mediated extraction pathway. Repressing the initial ERAD recognition step by inhibiting Grp94 enhances the functional surface expression of misfolding-prone α1(A322D) subunits, which causes autosomal dominant juvenile myoclonic epilepsy. This study clarifies a Grp94-mediated ERAD pathway for GABAA receptors, which provides a novel way to finely tune their function in physiological and pathophysiological conditions.

Combining valosin-containing protein (VCP) inhibition and suberanilohydroxamic acid (SAHA) treatment additively enhances the folding, trafficking, and function of epilepsy-associated γ-aminobutyric acid, type A (GABAA) receptors.

GABAA receptors are the primary inhibitory ion channels in the mammalian central nervous system. The A322D mutation in the α1 subunit results in its excessive endoplasmic reticulum-associated degradation at the expense of plasma membrane trafficking, leading to autosomal dominant juvenile myoclonic epilepsy. Presumably, valosin-containing protein (VCP)/p97 extracts misfolded subunits from the endoplasmic reticulum membrane to the cytosolic proteasome for degradation. Here we showed that inhibiting VCP using Eeyarestatin I reduces the endoplasmic reticulum-associated degradation of the α1(A322D) subunit without an apparent effect on its dynamin-1 dependent endocytosis and that this treatment enhances its trafficking. Furthermore, coapplication of Eeyarestatin I and suberanilohydroxamic acid, a known small molecule that promotes chaperone-assisted folding, yields an additive restoration of surface expression of α1(A322D) subunits in HEK293 cells and neuronal SH-SY5Y cells. Consequently, this combination significantly increases GABA-induced chloride currents in whole-cell patch clamping experiments than either chemical compound alone in HEK293 cells. Our findings suggest that VCP inhibition without stress induction, together with folding enhancement, represents a new strategy to restore proteostasis of misfolding-prone GABAA receptors and, therefore, a potential remedy for idiopathic epilepsy.

Docetaxel modulates the delayed rectifier potassium current (IK) and ATP-sensitive potassium current (IKATP) in human breast cancer cells.

Ion channel expression and activity may be affected during tumor development and cancer growth. Activation of potassium (K(+)) channels in human breast cancer cells is reported to be involved in cell cycle progression. In this study, we investigated the effects of docetaxel on the delayed rectifier potassium current (I K) and the ATP-sensitive potassium current (I KATP) in two human breast cancer cell lines, MCF-7 and MDA-MB-435S, using the whole-cell patch-clamp technique. Our results show that docetaxel inhibited the I K and I KATP in both cell lines in a dose-dependent manner. Compared with the control at a potential of +60 mV, treatment with docetaxel at doses of 0.1, 1, 5, and 10 µM significantly decreased the I K in MCF-7 cells by 16.1 ± 3.5, 30.2 ± 5.2, 42.5 ± 4.3, and 46.4 ± 9% (n = 5, P < 0.05), respectively and also decreased the I KATP at +50 mV. Similar results were observed in MDA-MB-435S cells. The G-V curves showed no significant changes after treatment of either MCF-7 or MDA-MB-435S cells with 10 µM docetaxel. The datas indicate that the possible mechanisms of I K and I KATP inhibition by docetaxel may be responsible for its effect on the proliferation of human breast cancer cells.

Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum.

Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric acid type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.

Identification of GABA(C) receptor protein homeostasis network components from three tandem mass spectrometry proteomics approaches.

γ-Amino butyric acid type C (GABA(C)) receptors inhibit neuronal firing primarily in retina. Maintenance of GABA(C) receptor protein homeostasis in cells is essential for its function. However, a systematic study of GABA(C) receptor protein homeostasis (proteostasis) network components is absent. Here coimmunoprecipitation of human GABA(C)-ρ1-receptor complexes was performed in HEK293 cells overexpressing ρ1 receptors. To enhance the coverage and reliability of identified proteins, immunoisolated ρ1-receptor complexes were subjected to three tandem mass spectrometry (MS)-based proteomic analyses, namely, gel-based tandem MS (GeLC-MS/MS), solution-based tandem MS (SoLC-MS/MS), and multidimensional protein identification technology (MudPIT). From the 107 identified proteins, we assembled GABA(C)-ρ1-receptor proteostasis network components, including proteins with protein folding, degradation, and trafficking functions. We studied representative individual ρ1-receptor-interacting proteins, including calnexin, a lectin chaperone that facilitates glycoprotein folding, and LMAN1, a glycoprotein trafficking receptor, and global effectors that regulate protein folding in cells based on bioinformatics analysis, including HSF1, a master regulator of the heat shock response, and XBP1, a key transcription factor of the unfolded protein response. Manipulating selected GABA(C) receptor proteostasis network components is a promising strategy to regulate GABA(C) receptor folding, trafficking, degradation and thus function to ameliorate related retinal diseases.

Cpt-cAMP activates human epithelial sodium channels via relieving self-inhibition.

External Na(+) self-inhibition is an intrinsic feature of epithelial sodium channels (ENaC). Cpt-cAMP regulates heterologous guinea pig but not rat αßγ ENaC in a ligand-gated manner. We hypothesized that cpt-cAMP may eliminate the self-inhibition of human ENaC thereby open channels. Regulation of self-inhibition by this compound in oocytes was analyzed using the two-electrode voltage clamp and Ussing chamber setups. External cpt-cAMP stimulated human but not rat and murine αßγ ENaC in a dose- and external Na(+) concentration-dependent fashion. Intriguingly, cpt-cAMP activated human δßγ more potently than αßγ channels, suggesting that structural diversity in ectoloop between human α, δ, and those ENaC of other species determines the stimulating effects of cpt-cAMP. Cpt-cAMP increased the ratio of stationary and maximal currents. Mutants having abolished self-inhibition (ß(ΔV348) and γ(H233R)) almost completely eliminated cpt-cAMP mediated activation of ENaC. On the other hand, mutants both enhancing self-inhibition and elevating cpt-cAMP sensitivity increased the stimulating effects of the compound. This compound, however, could not activate already fully opened channels, e.g., degenerin mutation (αß(S520C)γ) and the proteolytically cleaved ENaC by plasmin. Cpt-cAMP activated native ENaC to the same extent as that for heterologous ENaC in human lung epithelial cells. Our data demonstrate that cpt-cAMP, a broadly used PKA activator, stimulates human αßγ and δßγ ENaC channels by relieving self-inhibition.

Characterization of a novel splice variant of δ ENaC subunit in human lungs.

Salt absorption via apical epithelial sodium channels (ENaC) is a critical rate-limiting process in maintaining airway and lung lining fluid at the physiological level. δ ENaC (termed δ1 in this article) has been detected in human lung epithelial cells in addition to α, ß, and γ subunits (Ji HL, Su XF, Kedar S, Li J, Barbry P, Smith PR, Matalon S, Benos DJ. J Biol Chem 281: 8233-8241, 2006; Nie HG, Chen L, Han DY, Li J, Song WF, Wei SP, Fang XH, Gu X, Matalon S, Ji HL, J Physiol 587: 2663-2676, 2009) and may contribute to the differences in the biophysical properties of amiloride-inhibitable cation channels in pulmonary epithelial cells. Here we cloned a splicing variant of the δ1 ENaC, namely, δ2 ENaC in human bronchoalveolar epithelial cells (16HBEo). δ2 ENaC possesses 66 extra amino acids attached to the distal amino terminal tail of the δ1 ENaC. δ2 ENaC was expressed in both alveolar type I and II cells of human lungs as revealed by in situ hybridization and real-time RT-PCR. To characterize the biophysical and pharmacological features of the splicing variant, we injected Xenopus oocytes with human ENaC cRNAs and measured whole cell and single channel currents of δ1ßγ, δ2ßγ, and αßγ channels. Oocytes injected with δ2ßγ cRNAs exhibited whole cell currents significantly greater than those expressing δ1ßγ and αßγ channels. Single channel activity, unitary conductance, and open probability of δ2ßγ channels were significantly greater compared with δ1ßγ and αßγ channels. In addition, δ2ßγ and δ1ßγ channels displayed significant differences in apparent Na(+) affinity, dissociation constant for amiloride (K(i)(amil)), the EC(50) for capsazepine activation, and gating kinetics by protons. Channels comprising of this novel splice variant may contribute to the diversities of native epithelial Na(+) channels.

8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate-Na stimulates human alveolar fluid clearance by releasing external Na+ self-inhibition of epithelial Na+ channels.

Salt absorption via alveolar epithelial Na(+) channels (ENaC) is a critical step for maintaining an airspace free of flooding. Previously, we found that 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate-Na (CPT-cGMP) activated native and heterologous ENaC. To investigate the potential pharmacological relevance, we applied this compound intratracheally to human lungs and found that ex vivo alveolar fluid clearance was increased significantly. Furthermore, this compound eliminated self-inhibition in human lung H441 cells and in oocytes expressing human αßγ but not δßγ channels. To further elucidate this novel mechanism, we constructed mutants abolishing (ß(ΔV348) and γ(H233R)) or augmenting (α(Y458A) and γ(M432G)) self-inhibition. The mutants eliminating self-inhibition lost their responses to CPT-cGMP, whereas those enhancing self-inhibition facilitated the stimulatory effects of this compound. CPT-cGMP was unable to activate a high P(o) mutant (ß(S520C)) and plasmin proteolytically cleaved channels. Our data suggest that elimination of self-inhibition of αßγ ENaC may be a novel mechanism for CPT-cGMP to stimulate salt reabsorption in human lungs.

Proteostasis Regulators Restore Function of Epilepsy-Associated GABAA Receptors.

Proteostasis deficiency in mutated ion channels leads to a variety of ion channel diseases that are caused by excessive endoplasmic reticulum-associated degradation (ERAD) and inefficient membrane trafficking. We investigated proteostasis maintenance of γ-aminobutyric acid type A (GABAA) receptors, the primary mediators of neuronal inhibition in the mammalian central nervous system. We screened a structurally diverse, Food and Drug Administration-approved drug library and identified dinoprost (DNP) and dihydroergocristine (DHEC) as highly efficacious enhancers of surface expression of four epilepsy-causing trafficking-deficient mutant receptors. Furthermore, DNP and DHEC restore whole-cell and synaptic currents by incorporating mutated subunits into functional receptors. Mechanistic studies revealed that both drugs reduce subunit degradation by attenuating the Grp94/Hrd1/Sel1L/VCP-mediated ERAD pathway and enhance the subunit folding by promoting subunit interactions with major GABAA receptors-interacting chaperones, BiP and calnexin. In summary, we report that DNP and DHEC remodel the endoplasmic reticulum proteostasis network to restore the functional surface expression of mutant GABAA receptors.

8-pCPT-cGMP stimulates alphabetagamma-ENaC activity in oocytes as an external ligand requiring specific nucleotide moieties.

Epithelial sodium channels (ENaC) are regulated by protein kinase A, in addition to a broad spectrum of other protein kinases. It is not clear whether cGMP/PKG signaling might regulate ENaC activity. We examined the responses of alphabetagamma-ENaC channels expressed in Xenopus oocytes to 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), a cell-permeable cGMP analog. This compound stimulated human alphabetagamma-ENaC activity in a dose-dependent fashion, but cell-impermeable cGMP had no effect. Similar stimulatory effects of cGMP were observed in oocytes expressing either mouse or rat alphabetagamma-ENaC channels. The identical ion selectivity and amiloride sensitivity of the 8-pCPT-cGMP-activated currents to those of alphabetagamma-ENaC channels suggest that the cGMP-activated currents are associated with expressed ENaC. The PKGI activator Sp isomer of beta-phenyl-1,N(2)-etheno-8-bromo-cGMP did not elicit a rise in ENaC current and that the 8-pCPT-cGMP-induced activation of ENaC channels was blocked by incubating oocytes with a PKG inhibitor, but not with other cGMP-sensitive kinase inactivators for PKA, MEK, MAP, and PKC. Surprisingly, both site-directed mutation of putative consensus PKG phosphorylation sites and truncation of entire cytosolic NH(2)- and COOH-terminal tails did not alter the response to 8-pCPT-cGMP. The ENaC activity was activated to the same extent by 8-pCPT-cGMP in cells in which PKGII expression was knocked down using small interfering RNA. Analog to 8-CPT-cAMP, 8-pCPT-cGMP was capable of activating ENaC in the identical manner in cell-free outside-out patches. We conclude that the rapid upregulation of human alphabetagamma-ENaC activity in oocytes by external 8-pCPT-cGMP and 4-chlorothiolphenol-cAMP depends on the para-chlorophenylthiol and the hydroxy groups, and 8-pCPT-cGMP may serve as a novel ENaC ligand in addition to activating PKG signal.

Both N- and C-lobes of calmodulin are required for Ca2+-dependent regulations of CaV1.2 Ca2+ channels.

We investigated the concentration- and Ca(2+)-dependent effects of CaM mutants, CaM(12) and CaM(34), in which Ca(2+)-binding to its N- and C-lobes was eliminated, respectively, on the Ca(V)1.2 Ca(2+) channel by inside-out patch clamp in guinea-pig cardiomyocytes. Both CaM(12) and CaM(34) (0.7-10muM) applied with 3mM ATP produced channel activity after "rundown". Concentration-response curves were bell-shaped, similar to that for wild-type CaM. However, there was no obvious leftward shift of the curves by increasing [Ca(2+)], suggesting that both functional lobes of CaM were necessary for the Ca(2+)-dependent shift. However, channel activity induced by the CaM mutants showed Ca(2+)-dependent decrease, implying a Ca(2+) sensor existing besides CaM. These results suggest that both N- and C-lobes of CaM are required for the Ca(2+)-dependent regulations of Ca(V)1.2 Ca(2+) channels.

K+ channel openers restore verapamil-inhibited lung fluid resolution and transepithelial ion transport.

BACKGROUND: Lung epithelial Na+ channels (ENaC) are regulated by cell Ca2+ signal, which may contribute to calcium antagonist-induced noncardiogenic lung edema. Although K+ channel modulators regulate ENaC activity in normal lungs, the therapeutical relevance and the underlying mechanisms have not been completely explored. We hypothesized that K+ channel openers may restore calcium channel blocker-inhibited alveolar fluid clearance (AFC) by up-regulating both apical and basolateral ion transport. METHODS: Verapamil-induced depression of heterologously expressed human alphabetagamma ENaC in Xenopus oocytes, apical and basolateral ion transport in monolayers of human lung epithelial cells (H441), and in vivo alveolar fluid clearance were measured, respectively, using the two-electrode voltage clamp, Ussing chamber, and BSA protein assays. Ca2+ signal in H441 cells was analyzed using Fluo 4AM. RESULTS: The rate of in vivo AFC was reduced significantly (40.6+/-6.3% of control, P<0.05, n=12) in mice intratracheally administrated verapamil. KCa3.1 (1-EBIO) and KATP (minoxidil) channel openers significantly recovered AFC. In addition to short-circuit current (Isc) in intact H441 monolayers, both apical and basolateral Isc levels were reduced by verapamil in permeabilized monolayers. Moreover, verapamil significantly altered Ca2+ signal evoked by ionomycin in H441 cells. Depletion of cytosolic Ca2+ in alphabetagamma ENaC-expressing oocytes completely abolished verapamil-induced inhibition. Intriguingly, KV (pyrithione-Na), K Ca3.1 (1-EBIO), and KATP (minoxidil) channel openers almost completely restored the verapamil-induced decrease in Isc levels by diversely up-regulating apical and basolateral Na+ and K+ transport pathways. CONCLUSIONS: Our observations demonstrate that K+ channel openers are capable of rescuing reduced vectorial Na+ transport across lung epithelial cells with impaired Ca2+ signal.

Calmodulin- and Ca2+-dependent facilitation and inactivation of the Cav1.2 Ca2+ channels in guinea-pig ventricular myocytes.

The L-type Ca(2+) channel (Ca(V)1.2) shows clear Ca(2+)-dependent facilitation and inactivation. Here we have examined the effects of calmodulin (CaM) and Ca(2+) on Ca(2+) channel in guinea-pig ventricular myocytes in the inside-out patch mode, where rundown of the channels was controlled. At a free [Ca(2+)] of 0.1 microM, CaM (0.15, 0.7, 1.4, 2.1, 3.5, and 7.0 microM) + ATP (2.4 mM) induced channel activities of 27%, 98%, 142%, 222%, 65%, and 20% relative to the control activity, respectively, showing a bell-shaped relationship. Similar results were observed at a free [Ca(2+)] <0.01 microM or with a Ca(2+)-insensitive mutant, CaM(1234), suggesting that apoCaM may induce facilitation and inactivation of the channel activity. The bell-shaped curve of CaM was shifted to the lower concentration side with increasing [Ca(2+)]. A simple model for CaM- and Ca(2+)-dependent modulations of the channel activity, which involves two CaM-binding sites, was proposed. We suggest that both apoCaM and Ca(2+)/CaM can induce facilitation and inactivation of Ca(V)1.2 Ca(2+) channels and that the basic role of Ca(2+) is to accelerate CaM-dependent facilitation and inactivation.

Combined effects of green manure returning and addition of sewage sludge compost on plant growth and microorganism communities in gold tailings.

Remediation of gold tailings is often difficult due to their extremely barren nature and highly heavy metal concentrations. Returning green manure and applying sewage sludge compost have the beneficial effects of providing nutrients and improving the soil environment. The effects of green manure plants, alfalfa (Medicago sativa L.), ryegrass (Lolium perenne Linn.), and tall fescue (Festuca arundinacea), returning in situ on nutrients, bioavailability of trace metals, and community structure of microorganism in gold tailings amended with 0%, 5%, and 10% (weight/weight) sewage sludge compost on the top 4 cm of tailings (SSC-5, SSC-10) were investigated in a pot experiment. The results showed that the plant biomass and microbial biomass carbon in tailings significantly increased in the treatments with sewage sludge compost. The available N and available P and the availability of Zn decreased markedly with the returning of alfalfa and ryegrass. Moreover, through high-throughput sequencing, it was found that the returning of alfalfa had positive effects on the bacterial community richness but a negative impact on the fungal community richness. The microbial community diversity was reduced in the treatment without sewage sludge compost amendment and with alfalfa returning. However, the microbial community diversity was enriched in the treatment of alfalfa returning with sewage sludge compost. In each plant species, 9 dominant bacterial phyla and 10 dominant fungi phyla could be detected. Returning alfalfa green manure and applying sewage sludge compost led to a relative increase in the abundance of Proteobacteria and Ascomycota. These results demonstrated that returning alfalfa and applying sewage sludge compost could be effective in the ecological restoration of gold tailings.

Regulation of epithelial sodium channels by cGMP/PKGII.

Airway and alveolar fluid clearance is mainly governed by vectorial salt movement via apically located rate-limiting Na(+) channels (ENaC) and basolateral Na(+)/K(+)-ATPases. ENaC is regulated by a spectrum of protein kinases, i.e. protein kinase A (PKA), C (PKC), and G (PKG). However, the molecular mechanisms for the regulation of ENaC by cGMP/PKG remain to be elucidated. In the present study, we studied the pharmacological responses of native epithelial Na(+) channels in human Clara cells and human alphabetagammadelta ENaCs expressed in oocytes to cGMP. 8-pCPT-cGMP increased amiloride-sensitive short-circuit current (I(sc)) across H441 monolayers and heterologously expressed alphabetagammadelta ENaC activity in a dose-dependent manner. Similarly, 8-pCPT-cGMP (a PKGII activator) but not 8-Br-cGMP (a PKGI activator) increased amiloride-sensitive whole cell currents in H441 cells in the presence of CFTRinh-172 and diltiazem. In all cases, the cGMP-activated Na(+) channel activity was inhibited by Rp-8-pCPT-cGMP, a specific PKGII inhibitor. This was substantiated by the evidence that PKGII was the sole isoform expressed in H441 cells at the protein level. Importantly, intratracheal instillation of 8-pCPT-cGMP in BALB/c mice increased amiloride-sensitive alveolar fluid clearance by approximately 30%, consistent with the in vitro results. We therefore conclude that PKGII is an activator of lung epithelial Na(+) channels, which may expedite the resolution of oedematous fluid in alveolar sacs.

The distinct roles of calmodulin and calmodulin kinase II in the reversal of run-down of L-type Ca(2+) channels in guinea-pig ventricular myocytes.

In this study, we investigated the roles of calmodulin kinase II (CaMKII) and calmodulin (CaM) in the reversal of run-down of L-type Ca(2+) channels. Single Ca(2+)-channel activities in guinea-pig ventricular myocytes were recorded using the patch-clamp technique, and run-down of the channel activities was induced by inside-out patch formation in the basic internal solution. At 1 min after patch excision, 1 - 30 muM CaMKII mutant T286D (CaMKIIT286D), a constitutively active type of CaMKII, induced the Ca(2+)-channel activities to only 2% - 10% of that recorded in the cell-attached mode. However, in the presence of CaMKIIT286D, the time-dependent attenuation of CaM's effects in the reversal of run-down was abolished. A GST-fusion protein containing amino acids 1509 - 1789 of the C-terminal region of guinea-pig Cav1.2 (CT1) was prepared. In pull-down assays, CT1 treated with CaMKIIT286D showed a higher affinity for CaM compared with CT1 treated with phosphatase. We propose a model in which CaMKII-mediated phosphorylation of the channels regulates the binding of CaM to the channels in the reversal of run-down of L-type Ca(2+) channels.

Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors.

Biogenesis of membrane proteins is controlled by the protein homeostasis (proteostasis) network. We have been focusing on protein quality control of γ-aminobutyric acid type A (GABAA) receptors, the major inhibitory neurotransmitter-gated ion channels in mammalian central nervous system. Proteostasis deficiency in GABAA receptors causes loss of their surface expression and thus function on the plasma membrane, leading to epilepsy and other neurological diseases. One well-characterized example is the A322D mutation in the α1 subunit that causes its extensive misfolding and expedited degradation in the endoplasmic reticulum (ER), resulting in autosomal dominant juvenile myoclonic epilepsy. We aimed to correct misfolding of the α1(A322D) subunits in the ER as an approach to restore their functional surface expression. Here, we showed that application of BIX, a specific, potent ER resident HSP70 family protein BiP activator, significantly increases the surface expression of the mutant receptors in human HEK293T cells and neuronal SH-SY5Y cells. BIX attenuates the degradation of α1(A322D) and enhances their forward trafficking and function. Furthermore, because BiP is one major target of the two unfolded protein response (UPR) pathways: ATF6 and IRE1, we continued to demonstrate that modest activations of the ATF6 pathway and IRE1 pathway genetically enhance the plasma membrane trafficking of the α1(A322D) protein in HEK293T cells. Our results underlie the potential of regulating the ER proteostasis network to correct loss-of-function protein conformational diseases.

Calpastatin binds to a calmodulin-binding site of cardiac Cav1.2 Ca2+ channels.

Calpastatin is an endogenous inhibitor of calpain and composed of domain L (CS(L)), which interacts with the Cav1.2 channels, and four repetitive calpain inhibitory domains. We have previously found that CS(L) reprimes activity of the Cav1.2 channels in cell-free patches of cardiac myocytes [L.Y. Hao, A. Kameyama, S. Kuroki, J. Takano, E. Takano, M. Maki, M. Kameyama, Calpastatin domain L is involved in the regulation L-type of Ca2+ channels in guinea pig cardiac myocytes, Biochem. Biophys. Res. Commun. 279 (2000) 756-761; E. Minobe, L.Y. Hao, Z.A. Saud, J.J. Xu, A. Kameyama, M. Maki, K.K. Jewell, T. Parr, R.G. Bardsley, M. Kameyama, A region of calpastatin domain L that reprimes cardiac L-type Ca2+ channels, Biochem. Biophys. Res. Commun. 348 (2006) 288-294]. In this study, we explored the CS(L) interaction site in the Ca2+ channel by the pull-down method, using glutathione-S-transferase-fused fragment peptides of the Cav1.2 channel. CS(L) bound directly to a proximal region of the C-terminal tail of the channel, but not with the N-terminal tail, a distal region of the C-terminal tail or cytoplasmic loops between repeats I-II, II-III or III-IV. Furthermore IQ domain, but not EF-hand-like region or CB domain, in the C-terminal tail was found to bind with CS(L) in a partially Ca2+-dependent manner and in a probably competitive manner with calmodulin. These results suggest that CS(L) modulates Ca2+-channel activity through interacting with the calmodulin-binding site on the C-terminal tail of the Cav1.2 channel.

L-type Calcium Channel Blockers Enhance Trafficking and Function of Epilepsy-associated α1(D219N) Subunits of GABA(A) Receptors.

Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory ion channels in the mammalian central nervous system and play an essential role in regulating inhibition-excitation balance in neural circuits. The α1 subunit harboring the D219N mutation of GABAA receptors was reported to be retained in the endoplasmic reticulum (ER) and traffic inefficiently to the plasma membrane, leading to a loss of function of α1(D219N) subunits and thus idiopathic generalized epilepsy (IGE). We present the use of small molecule proteostasis regulators to enhance the forward trafficking of α1(D219N) subunits to restore their function. We showed that treatment with verapamil (4 µM, 24 h), an L-type calcium channel blocker, substantially increases the α1(D219N) subunit cell surface level in both HEK293 cells and neuronal SH-SY5Y cells and remarkably restores the GABA-induced maximal chloride current in HEK293 cells expressing α1(D219N)ß2γ2 receptors to a level that is comparable to wild type receptors. Our drug mechanism study revealed that verapamil treatment promotes the ER to Golgi trafficking of the α1(D219N) subunits post-translationally. To achieve that, verapamil treatment enhances the interaction between the α1(D219N) subunit and ß2 subunit and prevents the aggregation of the mutant protein by shifting the protein from the detergent-insoluble fractions to detergent-soluble fractions. By combining (35)S pulse-chase labeling and MG-132 inhibition experiments, we demonstrated that verapamil treatment does not inhibit the ER-associated degradation of the α1(D219N) subunit. In addition, its effect does not involve a dynamin-1 dependent endocytosis. To gain further mechanistic insight, we showed that verapamil increases the interaction between the mutant protein and calnexin and calreticulin, two major lectin chaperones in the ER. Moreover, calnexin binding promotes the forward trafficking of the mutant subunit. Taken together, our data indicate that verapamil treatment enhances the calnexin-assisted forward trafficking and subunit assembly, which leads to substantially enhanced functional surface expression of the mutant receptors. Since verapamil is an FDA-approved drug that crosses blood-brain barrier and has been used as an additional medication for some epilepsies, our findings suggest that verapamil holds great promise to be developed to ameliorate IGE resulting from α1(D219N) subunit trafficking deficiency.

SAHA enhances Proteostasis of epilepsy-associated α1(A322D)ß2γ2 GABA(A) receptors.

GABA(A) receptors are the primary inhibitory ion channels in the mammalian central nervous system. The A322D mutation in the α1 subunit of GABA(A) receptors is known to result in its degradation and reduce its cell surface expression, leading to loss of GABAA receptor function in autosomal dominant juvenile myoclonic epilepsy. Here, we show that SAHA, a FDA-approved drug, increases the transcription of the α1(A322D) subunit, enhances its folding and trafficking posttranslationally, increases its cell surface level, and restores the GABA-induced maximal current in HEK293 cells expressing α1(A322D)ß2γ2 receptors to 10% of that for wild-type receptors. To enhance the trafficking efficiency of the α1(A322D) subunit, SAHA increases the BiP protein level and the interaction between the α1(A322D) subunit and calnexin. SAHA is a drug that enhances epilepsy-associated GABAA receptor proteostasis.