The VAPB-PTPIP51 endoplasmic reticulum-mitochondria tethering proteins are present in neuronal synapses and regulate synaptic activity.

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of key neuronal functions. This signaling involves close physical contacts between the two organelles that are mediated by "tethering proteins" that function to recruit regions of ER to the mitochondrial surface. The ER protein, vesicle-associated membrane protein-associated protein B (VAPB) and the mitochondrial membrane protein, protein tyrosine phosphatase interacting protein-51 (PTPIP51), interact to form one such tether. Recently, damage to ER-mitochondria signaling involving disruption of the VAPB-PTPIP51 tethers has been linked to the pathogenic process in Parkinson's disease, fronto-temporal dementia (FTD) and related amyotrophic lateral sclerosis (ALS). Loss of neuronal synaptic function is a key feature of Parkinson's disease and FTD/ALS but the roles that ER-mitochondria signaling and the VAPB-PTPIP51 tethers play in synaptic function are not known. Here, we demonstrate that the VAPB-PTPIP51 tethers regulate synaptic activity. VAPB and PTPIP51 localise and form contacts at synapses, and stimulating neuronal activity increases ER-mitochondria contacts and the VAPB-PTPIP51 interaction. Moreover, siRNA loss of VAPB or PTPIP51 perturbs synaptic function and dendritic spine morphology. Our results reveal a new role for the VAPB-PTPIP51 tethers in neurons and suggest that damage to ER-mitochondria signaling contributes to synaptic dysfunction in Parkinson's disease and FTD/ALS.

Determinants of CREB degradation and KChIP2 gene transcription in cardiac memory.

BACKGROUND: Left ventricular pacing (LVP) to induce cardiac memory (CM) in dogs results in a decreased transient outward K current (I(to)) and reduced mRNA and protein of the I(to) channel accessory subunit, KChIP2. The KChIP2 decrease is attributed to a decrease in its transcription factor, cyclic adenosine monophosphate response element binding protein (CREB). OBJECTIVE: This study sought to determine the mechanisms responsible for the CREB decrease that is initiated by LVP. METHODS: CM was quantified as T-wave vector displacement in 18 LVP dogs. In 5 dogs, angiotensin II receptor blocker, saralasin, was infused before and during pacing. In 3 dogs, proteasomal inhibitor, lactacystin, was injected into the left anterior descending artery before LVP. Epicardial biopsy samples were taken before and after LVP. Neonatal rat cardiomyocytes (NRCM) were incubated with H(2)O(2) (50 micromol/l) for 1 hour with or without lactacystin. RESULTS: LVP significantly displaced the T-wave vector and was associated with increased lipid peroxidation and increased tissue angiotensin II levels. Saralasin prevented T-vector displacement and lipid peroxidation. CREB was significantly decreased after 2 hours of LVP and was comparably decreased in H(2)O(2)-treated NRCM. Lactacystin inhibited the CREB decrease in LVP dogs and H(2)O(2)-treated NRCM. LVP and H(2)O(2) both induced CREB ubiquitination, and the H(2)O(2)-induced CREB decrease was prevented by knocking down ubiquitin. CONCLUSION: LVP initiates myocardial angiotensin II production and reactive oxygen species synthesis, leading to CREB ubiquitination and its proteasomal degradation. This sequence of events would explain the pacing-induced reduction in KChIP2, and contribute to altered repolarization and the T-wave changes of cardiac memory.

Left versus right atrial difference in dominant frequency, K(+) channel transcripts, and fibrosis in patients developing atrial fibrillation after cardiac surgery.

BACKGROUND: The development of atrial fibrillation (AF) after cardiac surgery is associated with adverse outcomes; however, the mechanism(s) that trigger and maintain AF in these patients are unknown. OBJECTIVE: The purpose of this study was to test our hypothesis that postoperative AF is maintained by high-frequency sources in the left atrium (LA) resulting from ion channel and structural features that differ from the right atrium (RA). METHODS: Forty-four patients with no previous history of AF who underwent cardiac surgery consented to LA and RA biopsies. Histologic sections evaluated fatty infiltration, fibrosis, and iron deposition; quantitative reverse transcription-polymerase chain reaction (RT-PCR) assessed ion channel expression. In a subset of 27 patients, LA and RA unipolar recording leads were also placed. In patients who developed AF, the dominant frequency (DF) for each lead was calculated using fast Fourier transform. RESULTS: DFs during AF were LA 6.26 +/- 0.8 Hz, RA 4.56 +/- 0.7 Hz (P <.01). RT-PCR revealed LA-to-RA differences in mRNA abundance for Kir2.3 (1.8:1) and Kir3.4 (2.3:1). While LA fibrosis was greater in patients developing AF compared with those remaining in normal sinus rhythm (10.8% +/- 11% vs. 3.8% +/- 3.5%; P = .03), the amount of LA fibrosis inversely correlated with the LA DF. CONCLUSIONS: This is the first demonstration of LA-to-RA frequency differences during postoperative AF, which are associated with LA-to-RA differences in mRNA levels for potassium channel proteins and LA fibrosis. These results strongly suggest that sources of AF after cardiac surgery are located in the LA and are stabilized by LA fibrosis.

Proteomic analyses of native brain K(V)4.2 channel complexes.

Somatodendritic A-type (I(A)) voltage-gated K(+) (K(V)) channels are key regulators of neuronal excitability, functioning to control action potential waveforms, repetitive firing and the responses to synaptic inputs. Rapidly activating and inactivating somatodendritic I(A) channels are encoded by K(V)4 alpha subunits and accumulating evidence suggests that these channels function as components of macromolecular protein complexes. Mass spectrometry (MS)-based proteomic approaches were developed and exploited here to identify potential components and regulators of native brain K(V)4.2-encoded I(A) channel complexes. Using anti-K(V)4.2 specific antibodies, K(V)4.2 channel complexes were immunoprecipitated from adult wild type mouse brain. Parallel control experiments were performed on brain samples isolated from (K(V)4.2(-/-)) mice harboring a targeted disruption of the KCND2 (K(V)4.2) locus. Three proteomic strategies were employed: an in-gel approach, coupled to one-dimensional liquid chromatography-tandem MS (1D-LC-MS/MS), and two in-solution approaches, followed by 1D- or 2D-LC-MS/MS. The targeted in-gel 1D-LC-MS/MS analyses demonstrated the presence of the K(V)4 alpha subunits (K(V)4.2, K(V)4.3 and K(V)4.1) and the K(V)4 accessory, KChIP (KChIP1-4) and DPP (DPP6 and 10), proteins in native brain K(V)4.2 channel complexes. The more comprehensive, in-solution approach, coupled to 2D-LC-MS/MS, also called Multidimensional Protein Identification Technology (MudPIT), revealed that additional regulatory proteins, including the K(V) channel accessory subunit K(V)beta1, are also components of native brain K(V)4.2 channel complexes. Additional biochemical and functional approaches will be required to elucidate the physiological roles of these newly identified K(V)4 interacting proteins.

Complete 3D visualization of primate striosomes by KChIP1 immunostaining.

High-resolution 3D reconstruction and morphometric analysis of striosomes was carried out in macaque monkeys by using immunocytochemistry for the Kv4 potassium channel subunit potassium channel interacting protein 1 (KChIP1), a novel marker. The striosomes form a connected reticulum made up of two distinct planar sheets spanning several millimeters in the putamen, and long finger-like branches in the caudate nucleus and putamen. Although their spatial organization is variable, morphometric analysis of the striosomes, utilizing skeletonizations, reveals several quantitative invariant measures of striosome organization, including the following findings: 1) individual bifurcation-free striosome branches are 355 +/- 108.5 microm in diameter and 1,013 +/- 751 microm in length, and are both lognormally distributed; and 2) striosome branches exhibit three pronounced orientation preferences that are approximately orthogonal. The former finding suggests a fundamental anatomical and functional component of the striatum, whereas the latter indicates that striosomes are more lattice-like than their spatial variability suggests. The perceived variable spatial organization of the striosomes in primates belies many invariant features that may reflect striatal function, development, and pathophysiology.

Survey of the year 2005: literature on applications of isothermal titration calorimetry.

Isothermal titration calorimetry (ITC) can provide a full thermodynamic characterization of an interaction. Its usage does not suffer from constraints of molecular size, shape or chemical constitution. Neither is there any need for chemical modification or attachment to solid support. This ease of use has made it an invaluable instrumental resource and led to its appearance in many laboratories. Despite this, the value of the thermodynamic parameterization has, only quite recently, become widely appreciated. Although our understanding of the correlation between thermodynamic data and structural details continues to be somewhat naïve, a large number of publications have begun to improve the situation. In this overview of the literature for 2005, we have attempted to highlight works of interest and novelty. Furthermore, we draw attention to those works which we feel have provided a route to better analysis and increased our ability to understand the meaning of thermodynamic change on binding.