Modulation of Kv4-encoded K(+) currents in the mammalian myocardium by neuronal calcium sensor-1

Voltage-gated K(+) channels are multimeric proteins, consisting of four pore-forming alpha-subunits alone or in association with accessory subunits. Recently, for example, it was shown that the accessory Kv channel interacting proteins form complexes with Kv4 alpha-subunits and modulate Kv4 channel activity. The experiments reported here demonstrate that the neuronal calcium sensor protein-1 (NCS-1), another member of the recoverin-neuronal calcium sensor superfamily, is expressed in adult mouse ventricles and that NCS-1 co-immunoprecipitates with Kv4.3 from (adult mouse) ventricular extracts. In addition, co-expression studies in HEK-293 cells reveal that NCS-1 increases membrane expression of Kv4 alpha-subunits and functional Kv4-encoded K(+) current densities. Co-expression of NCS-1 also decreases the rate of inactivation of Kv4 alpha-subunit-encoded K(+) currents. In contrast to the pronounced effects of Kv channel interacting proteins on Kv4 channel gating, however, NCS-1 co-expression does not measurably affect the voltage dependence of steady-state inactivation or the rate of recovery from inactivation of Kv4-encoded K(+) currents. Taken together, these results suggest that NCS-1 is an accessory subunit of Kv4-encoded I(to,f) channels that functions to regulate I(to,f) density in the mammalian myocardium.     

A reassessment of the inhibitory capacity of human FKBP38 on calcineurin

The microbial peptidomacrolide FK506 affects many eukaryotic developmental and cell signaling programs via calcineurin inhibition. Prior formation of a complex between FK506 and intracellular FK506-binding proteins (FKBPs) is the precondition for the interaction with calcineurin. A puzzling difference has emerged between the mammalian multidomain protein hFKBP38 and other FKBPs. It was shown that hFKBP38 not only binds to calcineurin but also inhibits the protein phosphatase activity of calcineurin on its own [Shirane, M. and Nakayama, K.I. (2003) Nature Cell Biol. 5, 28-37]. Inherent calcineurin inhibition by hFKBP38 would completely eliminate the need for FK506 in controlling many signal transduction pathways. To address this issue, we have characterized the functional and physical interactions between calcineurin and hFKBP38. A recombinant hFKBP38 variant and endogenous hFKBP38 were tested both in vitro and in vivo. The proteins neither directly inhibited calcineurin activity nor affected NFAT reporter gene activity in SH-SY5Y and Jurkat cells. In addition, a direct physical interaction between calcineurin and hFKBP38 was not detected in co-immunoprecipitation experiments. However, hFKBP38 indirectly affected the subcellular distribution of calcineurin by interaction with typical calcineurin ligands, as exemplified by the anti-apoptotic protein Bcl-2. Our data suggest that hFKBP38 cannot substitute for the FKBP/FK506 complex in signaling pathways controlled by the protein phosphatase activity of calcineurin.     

Localization of synaptic proteins involved in neurosecretion in different membrane microdomains

A number of proteins and signalling molecules modulate voltage-gated calcium channel activity and neurosecretion. As recent findings have indicated the presence of Ca(v)2.1 (P/Q-type) channels and soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) in the cholesterol-enriched microdomains of neuroendocrine and neuronal cells, we investigated whether molecules known to modulate neurosecretion, such as the heterotrimeric G proteins and neuronal calcium sensor-1 (NCS-1), are also localized in these microdomains. After immuno-isolation, flotation gradients from Triton X-100-treated synaptosomal membranes revealed the presence of different detergent-resistant membranes (DRMs) containing proteins of the exocytic machinery (Ca(v)2.1 channels and SNAREs) or NCS-1; both DRM subtypes contained aliquots of heterotrimeric G protein subunits and phosphatidylinositol-4,5-bisphosphate. In line with the biochemical data, confocal imaging of immunolabelled membrane sheets revealed the localization of SNARE proteins and NCS-1 in different dot-like structures. This distribution was largely impaired by treatment with methyl-beta-cyclodextrin, thus suggesting the localization of all three proteins in cholesterol-dependent domains. Finally, bradykinin (which is known to activate the NCS-1 pathway) caused a significant increase in NCS-1 in the DRMs. These findings suggest that different membrane microdomains are involved in the spatial organization of the complex molecular network that converges on calcium channels and the secretory machinery.     

Targeting of calcineurin to an NFAT-like docking site is required for the calcium-dependent activation of the background K+ channel, TRESK

The two-pore domain K(+) channel, TRESK (TWIK-related spinal cord K(+) channel) is activated in response to the calcium signal by the calcium/calmodulin-dependent protein phosphatase, calcineurin. In the present study we report that calcineurin also interacts with TRESK via an NFAT-like docking site, in addition to its enzymatic action. In its intracellular loop, mouse TRESK possesses the amino acid sequence, PQIVID, which is similar to the calcineurin binding consensus motif, PXIXIT (where X denotes any amino acids), necessary for NFAT (nuclear factor of activated T cells) activation and nuclear translocation. Mutations of the PQIVID sequence of TRESK to PQIVIA, PQIVAD, or PQAVAD increasingly deteriorated the calcium-dependent activation in the listed order and correspondingly reduced the benzocaine sensitivity (a property discriminating activated channels from resting ones), when it was measured after the calcium signal in Xenopus oocytes. Microinjection of VIVIT peptide, designed to inhibit the NFAT-calcineurin interaction specifically, also eliminated TRESK activation. The intracellular loop of TRESK, expressed as a GST fusion protein, bound constitutively active calcineurin in vitro. PQAVAD mutation as well as addition of VIVIT peptide to the reaction abrogated this calcineurin binding. Wild type calcineurin was recruited to GST-TRESK-loop in the presence of calcium and calmodulin. These results indicate that the PQIVID sequence is a docking site for calcineurin, and its occupancy is required for the calcium-dependent regulation of TRESK. Immunosuppressive compounds, developed to target the NFAT binding site of calcineurin, are also expected to interfere with TRESK regulation, in addition to their desired effect on NFAT.     

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

We have developed a protocol to produce large quantities of high purity myristoylated and non-myristoylated neuronal calcium sensor 1 (NCS-1) protein. NCS-1 is a member of the neuronal calcium sensor (NCS) family and plays an important role in modulating G-protein signaling and exocytosis pathways in cells. Many of these functions are calcium-dependent and require NCS-1 to be modified with an N-terminal myristoyl moiety. In our system, a C-terminally 6x His-tagged variant of NCS-1 was co-expressed with yeast N-myristoyltransferase (NMT) in ZYP-5052 auto-induction media supplemented with sodium myristate (100-200 microM). With optimized growth conditions and a high capacity metal affinity purification scheme, >50mg of homogenous myristoylated NCS-1 is obtained from 1L of culture in a single step. The properties of the C-terminally tagged NCS-1 variants are indistinguishable from those reported for untagged NCS-1. Using this system, we have also isolated and characterized mutant NCS-1 proteins that have attenuated (NCS-1 E120Q) and abrogated (NCS-1 DeltaEF) ability to bind calcium. The large quantities of NCS-1 proteins isolated from small culture volumes of auto-inducible media will provide the necessary reagents for further biochemical and structural characterization. The affinity tag at the C-terminus of the protein provides a suitable reagent for easily identifying binding partners of the various NCS-1 constructs. Additionally, this method could be used to produce other recombinant proteins of the NCS family, and may be extended to express and isolate myristoylated variants of other proteins.     

Control of Myf5 activation in adult skeletal myonuclei requires ERK signalling

Myf5 plays a central role in determination of the myogenic lineage, yet the signalling pathways that control its activation remain unclear. In adult muscle, Myf5 is expressed in satellite cells and muscle spindles but not by myonuclei. However, Myf5 expression is activated in myonuclei in response to muscle denervation. This can be modelled in culture using Myf5nlacZ/+ mice, allowing signalling pathways controlling Myf5 to be readily examined. We found that mitogen-rich medium induces activation of the Myf5 locus through calcium, which interacts with calmodulin to promote calcineurin and calmodulin kinase. Calcineurin activates NFAT to control Myf5 activation, while p38/JNK activity prevents activation by this route. Calmodulin kinase however, acts predominately through ERK signalling to activate Myf5. Interestingly, we found that IGF-1 can substitute for mitogen-rich medium and activates Myf5 through calcium, PI3K and ERK pathways. Together these observations show that Myf5 activation in adult muscle is accomplished by a complex signalling pathway, and provides candidates that can be examined for their role in Myf5 regulation during development.     

The Dendritic Peptide Neurogranin Can Regulate a Calmodulin-Dependent Target

Abstract: Neurogranin, a peptide capable of binding the calcium-poor form of calmodulin, was tested in vitro for its ability to modulate a typical calmodulin target. The target employed was the calcium/calmodulin-dependent form of nitric oxide synthase, which is produced by several different types of neurons. Neurogranin for the study was purified from perchloric acid-soluble calf brain proteins by a combination of calmodulin-Sepharose affinity chromatography and reverse-phase HPLC. The protocol yielded highly purified neurogranin that was active in assays using purified nitric oxide synthase. The titration of the enzyme activity with neurogranin demonstrated a concentration-dependent effect of the peptide on enzyme activation. Subsequent analysis of the ability of increased calcium concentrations to activate the enzyme was performed in the presence of different amounts of neurogranin. The effect of neurogranin on the calcium-dependent activation of the enzyme was to depress enzyme activity in the range of 0.2 to ∼1 µ M calcium. Treatment of the neurogranin peptide with protein kinase C eliminated its inhibition on nitric oxide synthase activation. Treatment of the protein kinase C-phosphorylated peptide with calcineurin did not restore the ability of neurogranin to inhibit enzyme activity, whereas treatment with alkaline phosphatase did restore this ability. These results suggest that neurogranin may serve as a member of a unique class of endogenous calmodulin inhibitor that functions to regulate the activation of calmodulin-requiring targets in neurons.     

Inhibition of cyclic nucleotide phosphodiesterase and calcineurin by spermine, a calcium-independent calmodulin antagonist

Spermine binding to calmodulin and its effects on two calmodulin-dependent enzymes were studied. Spermine bound to dansylated calmodulin with an apparent Ki of 0.7 mM, and to native calmodulin with a Kd of 1.1 mM in equilibrium dialysis experiments. Its binding was found to be independent of calcium. Spermine inhibited calmodulin-activated cyclic nucleotide phosphodiesterase noncompetitively with respect to calcium (Ki = 1.1 mM). Calmodulin activation of calcineurin was inhibited at similar concentrations (Ki = 1.2 mM). Spermine had little effect on basal phosphodiesterase activity or nickel-activated calcineurin activity. Inhibition of both enzymes correlated well with spermine binding to dansylcalmodulin. These findings suggest that spermine might modulate calcium-dependent events in the cell by inactivation of calmodulin via a novel calcium-independent mechanism.