Manjot Bal

Ca2+/Calmodulin Disrupts AKAP79/150 Interactions with KCNQ (M-Type) K+ Channels

M-type channels are localized to neuronal, cardiovascular, and epithelial tissues, where they play critical roles in control of excitability and K+ transport, and are regulated by numerous receptors via Gq/11-mediated signals. One pathway shown for KCNQ2 and muscarinic receptors uses PKC, recruited to the channels by A-kinase anchoring protein (AKAP)79/150. As M-type channels can be variously composed of KCNQ1-5 subunits, and M current is known to be regulated by Ca2+/calmodulin (CaM) and PIP2, we probed the generality of AKAP79/150 actions among KCNQ1-5 channels, and the influence of Ca2+/CaM and PIP2 on AKAP79/150 actions. We first examined which KCNQ subunits are targeted by AKAP79 in Chinese hamster ovary (CHO) cells heterologously expressing KCNQ1-5 subunits and AKAP79, using fluorescence resonance energy transfer (FRET) under total internal reflection fluorescence (TIRF) microscopy, and patch-clamp analysis. Donor-dequenching FRET between CFP-tagged KCNQ1-5 and YFP-tagged AKAP79 revealed association of KCNQ2-5, but not KCNQ1, with AKAP79. In parallel with these results, CHO cells stably expressing M1 receptors studied under perforated patch-clamp showed cotransfection of AKAP79 to “sensitize” KCNQ2/3 heteromers and KCNQ2-5, but not KCNQ1, homomers to muscarinic inhibition, manifested by shifts in the dose–response relations to lower concentrations. The effect on KCNQ4 was abolished by the T553A mutation of the putative PKC phosphorylation site. We then probed the role of CaM and PIP2 in these AKAP79 actions. TIRF/FRET experiments revealed cotransfection of wild-type, but not dominant-negative (DN), CaM that cannot bind Ca2+, to disrupt the interaction of YFP-tagged AKAP791-153 with CFP-tagged KCNQ2-5. Tonic depletion of PIP2 by cotransfection of a PIP2 phosphatase had no effect, and sudden depletion of PIP2 did not delocalize GFP-tagged AKAP79 from the membrane. Finally, patch-clamp experiments showed cotransfection of wild-type, but not DN, CaM to prevent the AKAP79-mediated sensitization of KCNQ2/3 heteromers to muscarinic inhibition. Thus, AKAP79 acts on KCNQ2-5, but not KCNQ1-containing channels, with effects disrupted by calcified CaM, but not by PIP2 depletion.

Homomeric and heteromeric assembly of KCNQ (Kv7) K+ channels assayed by total internal reflection fluorescence/fluorescence resonance energy transfer and patch clamp analysis.

M-type K+ channels, consisting of KCNQ1–5 (Kv7.1–7.5) subunits, form a variety of homomeric and heteromeric channels. Whereas all the subunits can assemble into homomeric channels, the ability of the subunits to assemble into heteromultimers is highly variable. KCNQ3 is widely thought to co-assemble with several other KCNQ subtypes, whereas KCNQ1 and KCNQ2 do not. However, the existence of other subunit assemblies is not well studied. To systematically explore the heteromeric assembly of KCNQ channels in individual living cells, we performed fluorescence resonance energy transfer (FRET) between cyan fluorescent protein- and yellow fluorescent protein-tagged KCNQ subunits expressed in Chinese hamster ovary cells under total internal reflection fluorescence microscopy in which excitation light only penetrates several hundred nanometers into the cell, thus isolating membrane events. We found significant FRET between homomeric subunits as expected from their functional expression in heterologous expression systems. Also as expected from previous work, robust FRET was observed between KCNQ2 and KCNQ3. KCNQ3 and KCNQ4 also showed substantial FRET as did KCNQ4 and KCNQ5. To determine functional assembly of KCNQ4/KCNQ5 heteromers, we performed two types of experiments. In the first, we constructed a mutant tetraethylammonium ion-sensitive KCNQ4 subunit and tested its assembly with KCNQ5 by patch clamp analysis of the tetraethylammonium ion sensitivity of the resulting current; however, those data were not conclusive. In the second, we co-expressed a KCNQ4 (G285S) pore mutant with KCNQ5 and found the former to act as a dominant negative, suggesting co-assembly of the two types of subunits. These data confirm that among the allowed assembly conformations are KCNQ3/4 and KCNQ4/5 heteromers.

Use-Dependent AMPA Receptor Block Reveals Segregation of Spontaneous and Evoked Glutamatergic Neurotransmission

Earlier findings had suggested that spontaneous and evoked glutamate release activates non-overlapping populations of NMDA receptors. Here, we evaluated whether AMPA receptor populations activated by spontaneous and evoked release show a similar segregation. To track the receptors involved in spontaneous or evoked neurotransmission, we used a polyamine agent, philanthotoxin, that selectively blocks AMPA receptors lacking GluR2 subunits in a use-dependent manner. In hippocampal neurons obtained from GluR2-deficient mice, philanthotoxin application decreased AMPA-receptor-mediated spontaneous miniature EPSCs (AMPA-mEPSCs) down to 20% of their initial level within 5 min. In contrast, the same philanthotoxin application at rest decreased the subsequent AMPA-receptor-mediated evoked EPSCs (eEPSCs) only down to 80% of their initial value. A 10-min-long perfusion of philanthotoxin further decreased AMPA-eEPSC amplitudes to 60% of their initial magnitude, which remained substantially higher than the level of AMPA-mEPSC block achieved within 5 min. Finally, stimulation after removal of philanthotoxin resulted in reversal of AMPA-eEPSC block, verifying strict use dependence of philanthotoxin. These results support the notion that spontaneous and evoked neurotransmission activate distinct sets of AMPA receptors and bolster the hypothesis that synapses harbor separate microdomains of evoked and spontaneous signaling.

AKAP79/150 Signal Complexes in G-Protein Modulation of Neuronal Ion Channels

Voltage-gated M-type (KCNQ) K+ channels play critical roles in regulation of neuronal excitability. Previous work showed A-kinase-anchoring protein (AKAP)79/150-mediated protein kinase C (PKC) phosphorylation of M channels to be involved in M current (IM) suppression by muscarinic M1, but not bradykinin B2, receptors. In this study, we first explored whether purinergic and angiotensin suppression of IM in superior cervical ganglion (SCG) sympathetic neurons involves AKAP79/150. Transfection into rat SCG neurons of ΔA-AKAP79, which lacks the A domain necessary for PKC binding, or the absence of AKAP150 in AKAP150−/− mice, did not affect IM suppression by purinergic agonist or by bradykinin, but reduced IM suppression by muscarinic agonist and angiotensin II. Transfection of AKAP79, but not ΔA-AKAP79 or AKAP15, rescued suppression of IM by muscarinic receptors in AKAP150−/− neurons. We also tested association of AKAP79 with M1, B2, P2Y6, and AT1 receptors, and KCNQ2 and KCNQ3 channels, via Förster resonance energy transfer (FRET) on Chinese hamster ovary cells under total internal refection fluorescence microscopy, which revealed substantial FRET between AKAP79 and M1 or AT1 receptors, and with the channels, but only weak FRET with P2Y6 or B2 receptors. The involvement of AKAP79/150 in Gq/11-coupled muscarinic regulation of N- and L-type Ca2+ channels and by cAMP/protein kinase A was also studied. We found AKAP79/150 to not play a role in the former, but to be necessary for forskolin-induced upregulation of L-current. Thus, AKAP79/150 action correlates with the PIP2 (phosphatidylinositol 4,5-bisphosphate)-depletion mode of IM suppression, but does not generalize to Gq/11-mediated inhibition of N- or L-type Ca2+ channels.

Calmodulin binding to M‐type K+ channels assayed by TIRF/FRET in living cells

Calmodulin (CaM) binds to KCNQ2–4 channels within their carboxy termini, where it regulates channel function. The existing data have not resolved the Ca2+ dependence of the interaction between the channels and CaM. We performed glutathione S‐transferase (GST)‐pull‐down assays between purified KCNQ2–4 carboxy termini and CaM proteins to determine the Ca2+ dependence of the interaction in vitro. The assays showed substantial Ca2+ dependence of the interaction of the channels with wild‐type (WT) CaM, but not with dominant‐negative (DN) CaM. To demonstrate CaM–channel interactions in individual living cells, we performed fluorescence resonance energy transfer (FRET) between ECFP‐tagged KCNQ2–4 channels and EYFP‐tagged CaM expressed in CHO cells, performed under total internal reflection fluorescence (TIRF) microscopy, in which excitation light only penetrates several hundred nanometres into the cell, thus isolating membrane events. FRET was assayed between the channels and either WT or DN CaM, performed under conditions of normal [Ca2+]i, low [Ca2+]i or high [Ca2+]i induced by empirically optimized bathing solutions. The FRET data suggest a strong Ca2+ dependence for the interaction between WT CaM and KCNQ2, but less so for KCNQ3 and KCNQ4. FRET between all KCNQ2–4 channels and DN CaM was robust, and not significantly Ca2+ dependent. These data show interactions between CaM and KCNQ channels in living cells, and suggest that the interactions between KCNQ2–4 channels and CaM are likely to have Ca2+‐dependent and Ca2+‐independent components.

Klotho up-regulates renal calcium channel transient receptor potential vanilloid 5 (TRPV5) by intra- and extracellular N-glycosylation-dependent mechanisms.

Background: Klotho is a putative sialidase that modifies the extracellular N-glycan of TRPV5 to increase its surface expression. Results: Intracellular expression of Klotho increased TRPV5 via an N-glycosylation-dependent mechanism that requires the sialidase activity of Klotho. Conclusion: Klotho up-regulates TRPV5 surface abundance by intra- and extracellular mechanisms. Significance: We investigate a novel function of Klotho to regulate intracellular protein trafficking via enzymatic activity. The anti-aging protein Klotho is a type 1 membrane protein produced predominantly in the distal convoluted tubule. The ectodomain of Klotho is cleaved and secreted into the urine to regulate several ion channels and transporters. Secreted Klotho (sKL) up-regulates the TRPV5 calcium channel from the cell exterior by removing sialic acids from N-glycan of the channel and inhibiting its endocytosis. Because TRPV5 and Klotho coexpress in the distal convoluted tubule, we investigated whether Klotho regulates TRPV5 action from inside the cell. Whole-cell TRPV5-mediated channel activity was recorded in HEK cells coexpressing TRPV5 and sKL or membranous Klotho (mKL). Transfection of sKL, but not mKL, produced detectable Klotho protein in cell culture media. As for sKL, mKL increased TRPV5 current density. The role of sialidase activity of mKL acting inside is supported by findings that mutations of putative sialidase activity sites in sKL and mKL abrogated the regulation of TRPV5 but that the extracellular application of a sialidase inhibitor prevented the regulation of TRPV5 by sKL only. Mechanistically, coexpression with a dominant-negative dynamin II prevented the regulation of TRPV5 by sKL but not by mKL. In contrast, blocking forward trafficking by brefeldin A prevented the effect with mKL but not with sKL. Therefore, Klotho up-regulates TRPV5 from both the inside and outside of cells. The intracellular action of Klotho is likely due to enhanced forward trafficking of channel proteins, whereas the extracellular action is due to inhibition of endocytosis. Both effects involve putative Klotho sialidase activity. These effects of Klotho may play important roles regarding calcium reabsorption in the kidney.

Selective impact of MeCP2 and associated histone deacetylases on the dynamics of evoked excitatory neurotransmission

An imbalance between the strengths of excitatory and inhibitory synaptic inputs has been proposed as the cellular basis of autism and related neurodevelopmental disorders. Previous studies examining spontaneous levels of excitatory and inhibitory neurotransmission in the forebrain regions of methyl-CpG-binding protein 2 (Mecp2) mutant mice, models of the autism spectrum disorder Rett syndrome, have identified a decrease in excitatory drive, in some cases coupled with an increase in inhibitory synaptic strength, as a major source of this imbalance. Here, we reevaluated this question by examining the short-term dynamics of evoked neurotransmission between hippocampal neurons cultured from MeCP2 knockout mice and found a marked increase in evoked excitatory neurotransmission that is consistent with an increase in presynaptic release probability. This increase in evoked excitatory drive was not matched with alterations in evoked inhibitory neurotransmission. Moreover, we observed similar excitatory drive specific changes after the loss of key histone deacetylases (histone deacetylase 1 and 2) that form a complex with MeCP2 and mediate transcriptional regulation. These findings suggest a distinct role for MeCP2 and its cofactors in the regulation of evoked excitatory neurotransmission compared with their essential role in basal synaptic activity.

AKAP150-mediated TRPV1 sensitization is disrupted by calcium/calmodulin

BackgroundThe transient receptor potential vanilloid type1 (TRPV1) is expressed in nociceptive sensory neurons and is sensitive to phosphorylation. A-Kinase Anchoring Protein 79/150 (AKAP150) mediates phosphorylation of TRPV1 by Protein Kinases A and C, modulating channel activity. However, few studies have focused on the regulatory mechanisms that control AKAP150 association with TRPV1. In the present study, we identify a role for calcium/calmodulin in controlling AKAP150 association with, and sensitization of, TRPV1.ResultsIn trigeminal neurons, intracellular accumulation of calcium reduced AKAP150 association with TRPV1 in a manner sensitive to calmodulin antagonism. This was also observed in transfected Chinese hamster ovary (CHO) cells, providing a model for conducting molecular analysis of the association. In CHO cells, the deletion of the C-terminal calmodulin-binding site of TRPV1 resulted in greater association with AKAP150, and increased channel activity. Furthermore, the co-expression of wild-type calmodulin in CHOs significantly reduced TRPV1 association with AKAP150, as evidenced by total internal reflective fluorescence-fluorescence resonance energy transfer (TIRF-FRET) analysis and electrophysiology. Finally, dominant-negative calmodulin co-expression increased TRPV1 association with AKAP150 and increased basal and PKA-sensitized channel activity.Conclusionsthe results from these studies indicate that calcium/calmodulin interferes with the association of AKAP150 with TRPV1, potentially extending resensitization of the channel.

Mucin-1 Increases Renal TRPV5 Activity In Vitro, and Urinary Level Associates with Calcium Nephrolithiasis in Patients

Hypercalciuria is a major risk factor for nephrolithiasis. We previously reported that Uromodulin (UMOD) protects against nephrolithiasis by upregulating the renal calcium channel TRPV5. This channel is crucial for calcium reabsorption in the distal convoluted tubule (DCT). Recently, mutations in the gene encoding Mucin-1 (MUC1) were found to cause autosomal dominant tubulointerstitial kidney disease, the same disease caused by UMOD mutations. Because of the similarities between UMOD and MUC1 regarding associated disease phenotype, protein structure, and function as a cellular barrier, we examined whether urinary MUC1 also enhances TRPV5 channel activity and protects against nephrolithiasis. We established a semiquantitative assay for detecting MUC1 in human urine and found that, compared with controls (n=12), patients (n=12) with hypercalciuric nephrolithiasis had significantly decreased levels of urinary MUC1. Immunofluorescence showed MUC1 in the thick ascending limb, DCT, and collecting duct. Applying whole-cell patch-clamp recording of HEK cells, we found that wild-type but not disease mutant MUC1 increased TRPV5 activity by impairing dynamin-2- and caveolin-1-mediated endocytosis of TRPV5. Coimmunoprecipitation confirmed a physical interaction between TRPV5 and MUC1. However, MUC1 did not increase the activity of N-glycan-deficient TRPV5. MUC1 is characterized by variable number tandem repeats (VNTRs) that bind the lectin galectin-3; galectin-3 siRNA but not galectin-1 siRNA prevented MUC1-induced upregulation of TRPV5 activity. Additionally, MUC1 lacking VNTRs did not increase TRPV5 activity. Our results suggest that MUC1 forms a lattice with the N-glycan of TRPV5 via galectin-3, which impairs TRPV5 endocytosis and increases urinary calcium reabsorption.

Sphingomimetic multiple sclerosis drug FTY720 activates vesicular synaptobrevin and augments neuroendocrine secretion

Neurotransmission and secretion of hormones involve a sequence of protein/lipid interactions with lipid turnover impacting on vesicle trafficking and ultimately fusion of secretory vesicles with the plasma membrane. We previously demonstrated that sphingosine, a sphingolipid metabolite, promotes formation of the SNARE complex required for membrane fusion and also increases the rate of exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and in hippocampal neurons. Recently a fungi-derived sphingosine homologue, FTY720, has been approved for treatment of multiple sclerosis. In its non-phosphorylated form FTY720 accumulates in the central nervous system, reaching high levels which could affect neuronal function. Considering close structural similarity of sphingosine and FTY720 we investigated whether FTY720 has an effect on regulated exocytosis. Our data demonstrate that FTY720 can activate vesicular synaptobrevin for SNARE complex formation and enhance exocytosis in neuroendocrine cells and neurons.