The C terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore

Bax inhibitor-1 (BI-1) is a multitransmembrane domain-spanning endoplasmic reticulum (ER)-located protein that is evolutionarily conserved and protects against apoptosis and ER stress. Furthermore, BI-1 is proposed to modulate ER Ca(2+) homeostasis by acting as a Ca(2+)-leak channel. Based on experimental determination of the BI-1 topology, we propose that its C terminus forms a Ca(2+) pore responsible for its Ca(2+)-leak properties. We utilized a set of C-terminal peptides to screen for Ca(2+) leak activity in unidirectional (45)Ca(2+)-flux experiments and identified an α-helical 20-amino acid peptide causing Ca(2+) leak from the ER. The Ca(2+) leak was independent of endogenous ER Ca(2+)-release channels or other Ca(2+)-leak mechanisms, namely translocons and presenilins. The Ca(2+)-permeating property of the peptide was confirmed in lipid-bilayer experiments. Using mutant peptides, we identified critical residues responsible for the Ca(2+)-leak properties of this BI-1 peptide, including a series of critical negatively charged aspartate residues. Using peptides corresponding to the equivalent BI-1 domain from various organisms, we found that the Ca(2+)-leak properties were conserved among animal, but not plant and yeast orthologs. By mutating one of the critical aspartate residues in the proposed Ca(2+)-channel pore in full-length BI-1, we found that Asp-213 was essential for BI-1-dependent ER Ca(2+) leak. Thus, we elucidated residues critically important for BI-1-mediated Ca(2+) leak and its potential channel pore. Remarkably, one of these residues was not conserved among plant and yeast BI-1 orthologs, indicating that the ER Ca(2+)-leak properties of BI-1 are an added function during evolution.     

Huntingtin and huntingtin-associated protein 1 influence neuronal calcium signaling mediated by inositol-(1,4,5) triphosphate receptor type 1

Huntington's disease (HD) is caused by polyglutamine expansion (exp) in huntingtin (Htt). The type 1 inositol (1,4,5)-triphosphate receptor (InsP3R1) is an intracellular calcium (Ca2+) release channel that plays an important role in neuronal function. In a yeast two-hybrid screen with the InsP3R1 carboxy terminus, we isolated Htt-associated protein-1A (HAP1A). We show that an InsP3R1-HAP1A-Htt ternary complex is formed in vitro and in vivo. In planar lipid bilayer reconstitution experiments, InsP3R1 activation by InsP3 is sensitized by Httexp, but not by normal Htt. Transfection of full-length Httexp or caspase-resistant Httexp, but not normal Htt, into medium spiny striatal neurons faciliates Ca2+ release in response to threshold concentrations of the selective mGluR1/5 agonist 3,5-DHPG. Our findings identify a novel molecular link between Htt and InsP3R1-mediated neuronal Ca2+ signaling and provide an explanation for the derangement of cytosolic Ca2+ signaling in HD patients and mouse models.     

Deranged neuronal calcium signaling and Huntington disease

Huntington disease (HD) is an autosomal-dominant neurodegenerative disorder that primarily affects medium spiny striatal neurons (MSN). HD is caused by polyglutamine (polyQ) expansion (exp) in the amino-terminal region of a protein huntingtin (Htt). The connection between polyQ expansion in Httexp and MSN neurodegeneration remains elusive. Here we discuss recent data that link polyQ expansion in Httexp and deranged Ca2+ signaling in MSN neurons. Experimental evidence indicates that (1) Ca2+ homeostasis is abnormal in mitochondria isolated from lymphoblasts of HD patients and from brains of the YAC72 HD mouse model; (2) Httexp leads to potentiation of NR1/NR2B NMDA receptor activity in heterologous expression systems and in MSN from YAC72 HD mouse model; and (3) Httexp binds to the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) carboxy-terminus and causes sensitization of InsP3R1 to activation by InsP3 in planar lipid bilayers and in MSN. Based on these results we propose that Httexp-induced cytosolic and mitochondrial Ca2+ overload of MSN plays an important role in the pathogenesis of HD and that Ca2+ signaling blockers may play a beneficial role in treatment of HD.     

Association of type 1 inositol 1,4,5-trisphosphate receptor with AKAP9 (Yotiao) and protein kinase A

Inositol 1,4,5-trisphosphate receptors (InsP(3)R) play a key role in intracellular calcium (Ca(2+)) signaling. Three InsP(3)R isoforms are expressed in mammals. Type 1 InsP(3)R (InsP(3)R1) is a predominant neuronal isoform. Neuronal InsP(3)R1 is one of the major substrates of protein kinase A (PKA) phosphorylation. In our previous study (Tang, T. S., Tu, H., Wang, Z., and Bezprozvanny, I. (2003) J. Neurosci. 23, 403-415) we discovered a direct association between InsP(3)R1 and protein phosphatase 1 alpha (PP1 alpha). In functional experiments we demonstrated that phosphorylation by PKA activates InsP(3)R1 and that dephosphorylation by PP1 alpha inhibits InsP(3)R1. To extend these findings, here we investigated the possibility of InsP(3)R1-PKA association. In a series of biochemical experiments we demonstrate the following findings. 1) InsP(3)R1 and PKA associate in the brain. 2) InsP(3)R1-PKA association is mediated by the AKAP9 (Yotiao) multi-functional PKA anchoring protein. 3) InsP(3)R1-AKAP9 association is mediated via the leucine/isoleucine zipper (LIZ) motif in the InsP(3)R1 coupling domain and the fourth LIZ motif in AKAP9. 4) The InsP(3)R association with AKAP9 is specific for type 1 InsP(3)R. 5) Both the SII(+) and the SII(-) coupling domain splice variants of InsP(3)R1 bind to AKAP9. 6) Binding to AKAP9 promotes association of neuronal InsP(3)R1 with the NR1 NMDA receptor; and 7) neuronal InsP(3)R1 associate with PP1 directly via carboxy-terminus and indirectly via AKAP9. The obtained results advance our understanding of cross-talk between cAMP and InsP(3)/Ca(2+) signaling pathways in the brain.     

Functional properties of endogenous receptor- and store-operated calcium influx channels in HEK293 cells

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.     

Functional characterization of the type 1 inositol 1,4,5-trisphosphate receptor coupling domain SII(+/-) splice variants and the Opisthotonos mutant form

The type 1 inositol (1,4,5)-trisphosphate receptor (InsP3R1) plays a critical role in Ca2+ signaling in cells. Neuronal and nonneuronal isoforms of the InsP3R1 differ by alternative splicing in the coupling domain of the InsP3R1 (SII site). Deletion of 107 amino acids from the coupling domain of the InsP3R1 results in epileptic-like behaviors in opisthotonos (opt) spontaneous mouse mutant. Using Spodoptera frugiperda cells expression system, we compared single-channel behavior of recombinant InsP3R1-SII(+), InsP3R1-SII(-), and InsP3R1-opt channels in planar lipid bilayers. The main results of our study are: 1) the InsP3R1-SII(-) has a higher conductance (94 pS) and the InsP3R1-opt has a lower conductance (64 pS) than the InsP3R1-SII(+) (81 pS); 2) the bell-shaped Ca2+-dependence peaks at 200-300 nM Ca2+ for all three InsP3R1 isoforms; 3) the bell-shaped Ca2+-dependence is wider for the InsP3R1-SII(+) and narrower for the InsP3R1-SII(-) and InsP3R1-opt; 4) the apparent affinity for ATP is sixfold lower for the InsP3R1-SII(-) (1.4 mM) and 20-fold lower for the InsP3R1-opt (5.3 mM) than for the InsP3R1-SII(+) (0.24 mM); 5) the InsP3R1-SII(-) is approximately twofold more active than the InsP3R1-SII(+) in the absence of ATP. Obtained results provide novel information about the molecular determinants of the InsP3R1 function.     

Activation of the calcium release channel (ryanodine receptor) by heparin and other polyanions is calcium dependent.

Heparin has been used as a potent competitive inhibitor of inositol 1,4,5-trisphosphate (IP3)-binding to IP3 receptors and to block IP3-gated calcium channels in bilayer experiments. In contrast to the effect on the IP3-gated channel, heparin (0.1-1 micrograms/ml) opened the Ca release channel (ryanodine receptor). Other polyanions such as pentosan polysulfate and polyvinyl sulfate also activated the Ca release channel. The effect of polyanions on the Ca release channel was Ca dependent. Polyanion addition activated the Ca release channel when free Ca was > 80 nM, but was ineffective when free Ca was < 20 nM. The level of channel activation could be altered by manipulating the free Ca concentration. These results suggest that the polyanions act by increasing the local concentration of Ca near regulatory sites on the channel complex. As most cells have both types of intracellular channels, the opposite effects of the polyanions on the two channel types suggests that addition of polyanions to intact cells may produce multiple effects.     

Single-Channel Properties of Inositol (1,4,5)-Trisphosphate Receptor Heterologously Expressed in HEK-293 Cells

The inositol (1,4,5)-trisphosphate receptor (InsP₃R) mediates Ca²⁺ release from intracellular stores in response to generation of second messenger InsP₃. InsP₃R was biochemically purified and cloned, and functional properties of native InsP₃-gated Ca²⁺ channels were extensively studied. However, further studies of InsP₃R are obstructed by the lack of a convenient functional assay of expressed InsP₃R activity. To establish a functional assay of recombinant InsP₃R activity, transient heterologous expression of neuronal rat InsP₃R cDNA (InsP₃R-I, SI− SII+ splice variant) in HEK-293 cells was combined with the planar lipid bilayer reconstitution experiments. Recombinant InsP₃R retained specific InsP₃ binding properties (K _d = 60 nM InsP₃) and were specifically recognized by anti–InsP₃R-I rabbit polyclonal antibody. Density of expressed InsP₃R-I was at least 20-fold above endogenous InsP₃R background and only 2–3-fold lower than InsP₃R density in rat cerebellar microsomes. When incorporated into planar lipid bilayers, the recombinant InsP₃R formed a functional InsP₃-gated Ca²⁺ channel with 80 pS conductance using 50 mM Ba²⁺ as a current carrier. Mean open time of recombinant InsP₃-gated channels was 3.0 ms; closed dwell time distribution was double exponential and characterized by short (18 ms) and long (130 ms) time constants. Overall, gating and conductance properties of recombinant neuronal rat InsP₃R-I were very similar to properties of native rat cerebellar InsP₃R recorded in identical experimental conditions. Recombinant InsP₃R also retained bell-shaped dependence on cytosolic Ca²⁺ concentration and allosteric modulation by ATP, similar to native cerebellar InsP₃R. The following conclusions are drawn from these results. (a) Rat neuronal InsP₃R-I cDNA encodes a protein that is either sufficient to produce InsP₃-gated channel with functional properties identical to the properties of native rat cerebellar InsP₃R, or it is able to form a functional InsP₃-gated channel by forming a complex with proteins endogenously expressed in HEK-293 cells. (b) Successful functional expression of InsP₃R in a heterologous expression system provides an opportunity for future detailed structure–function characterization of this vital protein.     

Association of neuronal calcium channels with modular adaptor proteins.

Presynaptic voltage-gated calcium (Ca(2+)) channels mediate Ca(2+) influx into the presynaptic terminal that triggers synaptic vesicle fusion and neurotransmitter release. The immediate proximity of Ca(2+) channels to the synaptic vesicle release apparatus is critical for rapid and efficient synaptic transmission. In a series of biochemical experiments, we demonstrate a specific association of the cytosolic carboxyl terminus of the N-type Ca(2+) channel pore-forming alpha(1B) subunit with the modular adaptor proteins Mint1 and CASK. The carboxyl termini of alpha(1B) bind to the first PDZ domain of Mint1 (Mint1-1). The proline-rich region present in the carboxyl termini of alpha(1B) binds to the SH3 domain of CASK. Mint1-1 is specific for the E/D-X-W-C/S-COOH consensus, which defines a novel class of PDZ domains (class III). The Mint1-1 PDZ domain-binding motif is present only in the "long" carboxyl-terminal splice variants of N-type (alpha(1B)) and P/Q-type (alpha(1A)) Ca(2+) channels, but not in R-type (alpha(1E)) or L-type (alpha(1C)) Ca(2+) channels. Our results directly link presynaptic Ca(2+) channels to a macromolecular complex formed by modular adaptor proteins at synaptic junction and advance our understanding of coupling between cell adhesion and synaptic vesicle exocytosis.