Cyclic AMP-vepenvent protein kinase phosphorylates residues in the C-terminal domain of the cardiac L-type calcium channel α1 subunit

AbstractThe molecular basis of the regulation of cardiac L-type calcium channel activity by cAMP-vepenvent protein kinase (cA-PK) remains unclear. Direct cA-PK-vepenvent phosphorylation of the bovine ventricular α1 subunit in vitro has been vemonstrated in microsomal membranes, vetergent extracts and partially purified (+)-[3H]PN 200-100 receptor preparations. Two 32P-labelled phosphopeptives, herived from cyanogen bromive cleavage, of 4.7 and 9.5 kDa were immunoprecipitated specifically by site-directed antibodies against the rabbit cardiac α1 subunit amino acid sequences 1602–1616 and 1681–1694, respectively, consistent with phosphorylation at the cA-PK consensus sites at Ser1627 and Ser1700. No phosphopeptive products consistent with phosphorylation at three other C-terminal cA-PK consensus phosphorylation sites (Ser1575, Ser1848 and Ser1928) were iventified using similar procedures suggesting that these sites are poor substrates for this kinase. Ser1627 and Ser1700 may represent sites of cA-PK phosphorylation involved in the physiological regulation of cardiac L-type calcium channel function

Voltage-dependent calcium channel β-subunits in combination with α1 subunits, have a GTPase activating effect to promote the hydrolysis of GTP by Gαo in rat frontal cortex

AbstractThe dihydropyridine-sensitive calcium channel agonist (−)-BayK 8644 was found to produce an enhancement of the intrinsic hydrolysis of GTP by Go in rat frontal cortex membranes. An anti-calcium channel β-subunit antiserum abolished the (−)-BayK 8644-stimulated hydrolysis of GTP by Go and reduced the dihydropyridine binding capacity of the cortical membranes. A peptide which mimics the β-subunit binding domain of the calcium channel complex, also attenuated (−)-BayK 8644 activation of GTPase. This study suggests that the calcium channel β-subunit is the principal component of the channel complex involved in linking dihydropyridine agonist binding to enhanced hydrolysis of GTP by Go. This may be a mechanism by which calcium channels can normally act to limit the duration of a G-protein modulatory signal

Anti-peptide antibodies as probes of the structure, function and distribution of Ca2+ channels

Fifteen peptides were synthesized corresponding to sequences in the primary structure of the a1 subunits of both the class A and class D rat brain, and of the a1 and a2 subunits of the rabbit skeletal muscle L-type Ca2+ channel. Conjugates of these peptides to various carrier proteins were used to produce polyclonal antibodies in rabbits. Each peptide-specific antibody was affinity purified and used to probe the structure, function and distribution of these channels. Nine of the antibodies identified (four strongly) their intact denatured polypeptide in t-tubules. The rat brain class A- specific antibody reacted strongly with a denatured polypeptide in skeletal muscle t- tubules, while only antibody N, raised against a sequence in a2, bound to its denatured polypeptide in brain membranes. Eight antibodies bound (four strongly) to the L-type Ca2+ channel in situ, in rabbit, rat, mouse and human skeletal muscle. One antibody reacted strongly, and three weakly with the corresponding channel in situ. in cardiac muscle cryosections from rabbit, rat and pig. Nine of the antibodies recognized (five very clearly) the intact, native, L-type channel purified from rabbit skeletal muscle. None of these antibodies showed any inhibition of binding of nitrendipine to the L-type Ca2+ channel in t-tubule membranes. The reactivity of the antibodies with the intact denatured channel polypeptides was examined in Western blots with rabbit and rat skeletal muscle t-tubules and brain membranes. In situ, identification of antibody binding was carried out using fluorescence immunocytochemistry in unfixed cryosections of skeletal muscle from rabbit, rat, mouse and human tissue and of cardiac muscle from rabbit, rat and pig. Antibody interaction with the native channel structure was studied by ELISA with the L-type channel following its purification from rabbit skeletal muscle. Inhibition of binding of either nitrendipine or D888 to the channel by the antibodies was assayed using radiolabelled ligand and t-tubule membranes which had been incubated with either test or control antibody. These antibodies have been used to confirm the predicted high sequence homology between rabbit skeletal muscle L-type Ca2+ channel a subunits and those found in rabbit, rat, human and mouse skeletal muscle, and in rat and porcine cardiac muscle, whose sequences are unknown. The IS4 domain, and peptides located on predicted intracellular loops and in the C-terminal region appeared to be exposed in the native α1 subunit. However, binding of dihydropyridines or phenylalkylamines to α1 was not affected by antibodies bound to any of the exposed α1 domains. Some β-sheet structure was revealed for a synthetic peptide, corresponding to the C-terminal α1(1390-1437) domain, in aqueous buffer using FTIR spectroscopic studies, L-type Ca2+ α polypeptides were located primarily to the transverse-tubule system of both skeletal and cardiac muscle. The relative abundance of the channel α2 subunit in rat brain membranes, was found to be not more than 10-30-fold less than that found in rat skeletal muscle transverse tubule membranes. The observed exposure of the IS4 domain in native a1 supports the model of the pore structure of voltage-gated ion channels, having both S2 and S4 as pore forming sequences, with the channel lined by the S5-S6 loop. Part of the loop between domain II and III in skeletal muscle α1, which has a role in excitation- contraction coupling is possibly inaccessible to antibody in situ, but becomes exposed during channel purification. A polypeptide, possibly a neuronal-type channel subunit was identified in t-tubules by the rat brain class A-specific antibody

Trafficking Kinesin Protein (TRAK)-mediated Transport of Mitochondria in Axons of Hippocampal Neurons*

In neurons, the proper distribution of mitochondria is essential because of a requirement for high energy and calcium buffering during synaptic neurotransmission. The efficient, regulated transport of mitochondria along axons to synapses is therefore crucial for maintaining function. The trafficking kinesin protein (TRAK)/Milton family of proteins comprises kinesin adaptors that have been implicated in the neuronal trafficking of mitochondria via their association with the mitochondrial protein Miro and kinesin motors. In this study, we used gene silencing by targeted shRNAi and dominant negative approaches in conjunction with live imaging to investigate the contribution of endogenous TRAKs, TRAK1 and TRAK2, to the transport of mitochondria in axons of hippocampal pyramidal neurons. We report that both strategies resulted in impairing mitochondrial mobility in axonal processes. Differences were apparent in terms of the contribution of TRAK1 and TRAK2 to this transport because knockdown of TRAK1 but not TRAK2 impaired mitochondrial mobility, yet both TRAK1 and TRAK2 were shown to rescue transport impaired by TRAK1 gene knock-out. Thus, we demonstrate for the first time the pivotal contribution of the endogenous TRAK family of kinesin adaptors to the regulation of mitochondrial mobility

Identification, molecular cloning, and characterization of a novel GABAA receptor-associated protein, GRIF-1

A novel 913-amino acid protein, gamma-aminobutyric acid type A (GABA(A)) receptor interacting factor-1 (GRIF-1), has been cloned and identified as a GABA(A) receptor-associated protein by virtue of its specific interaction with the GABA(A) receptor beta 2 subunit intracellular loop in a yeast two-hybrid assay. GRIF-1 has no homology with proteins of known function, but it is the rat orthologue of the human ALS2CR3/KIAA0549 gene. GRIF-1 is expressed as two alternative splice forms, GRIF-1a and a C-terminally truncated form, GRIF-1b. GRIF-1 mRNA has a wide distribution with a major transcript size of 6.2 kb. GRIF-1a protein is only expressed in excitable tissues, i.e. brain, heart, and skeletal muscle major immunoreactive bands of M(r) approximately 115 and 106 kDa and, in muscle and heart only, an additional 88-kDa species. When expressed in human embryonic kidney 293 cells, GRIF-1a yielded three immunoreactive bands with M(r) approximately 115, 106, and 98 kDa. Co-expression of GRIF-1a and alpha 1 beta 2 gamma 2 GABA(A) receptors in mammalian cells revealed some co-localization in the cell cytoplasm. Anti-FLAG-agarose specifically precipitated GRIF-1(FLAG) and GABA(A) receptor beta 2 subunits from human embryonic kidney 293 cells co-transfected with GRIF-1a(FLAG) and beta 2 subunit clones. Further, immobilized GRIF-1-(8-633) specifically precipitated in vitro GABA(A) receptor alpha 1 and beta 2 subunit immunoreactivities from detergent extracts of adult rat brain. The respective GABA(A) receptor beta 2 subunit/GRIF-1 binding domains were mapped using the yeast two-hybrid reporter gene assays. A possible role for GRIF-1 as a GABA(A) receptor beta 2 subunit trafficking factor is proposed

Differential activity-dependent regulation of the lateral mobilities of AMPA and NMDA receptors

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