Population patch clamp improves data consistency and success rates in the measurement of ionic currents

Present whole-cell patch-clamp methodology has only moderate consistency and throughput, rendering impractical functional measurements on large numbers of ion channel ligands or on large numbers of unknown or mutant channel genes. In the population patch clamp (PPC) described herein, a single voltage-clamp amplifier sums the whole-cell currents from multiple cells at once, each sealed to a separate aperture in a planar substrate well. The resulting ensemble currents are more consistent from well to well, and the success rate for each recording attempt is >95%. The PPC was implemented by modifying the PatchPlate substrate and amplifiers in the IonWorks patch-clamp instrument. The increased data consistency and likelihood of a successful recording in each well, combined with 384-well measurements in parallel, allow the direct electrophysiological recording of thousands of ensemble ionic currents per day. Therapeutic groups in drug discovery programs require this order of throughput to screen directed compound libraries against ion channel targets. The potential for studying the function of large numbers of ion channel mutants may be realized with the technique. The procedure incorporates subtraction methods that correct for expected distortions and also reliably produces data that agree with previous patch-clamp studies.     

Population patch clamp electrophysiology: a breakthrough technology for ion channel screening

Population patch clamp (PPC) is a novel high throughput planar array electrophysiology technique that allows ionic currents to be recorded from populations of cells under voltage clamp. For the drug discovery pharmacologist, PPC promises greater speed and precision than existing methods for screening compounds at voltage-gated ion channel targets. Moreover, certain constitutively active or slow-ligand gated channels that have hitherto proved challenging to screen with planar array electrophysiology (e.g. SK/IK channels) are now more accessible. In this article we review early findings using PPC and provide a perspective on its likely impact on ion channel drug discovery. To support this, we include some new data on ion channel assay duplexing and on modulator assays, approaches that have thus far not been described.     

Probing whole cell currents in high-frequency electrical fields: identification of thermal effects

An open-end coaxial probe is combined with a planar patch-clamp system to apply electric fields with GHz frequencies during conventional patch-clamp measurements. The combination of pulsed microwave irradiation and lock-in detection allows for the separation of fast and slow effects and hence facilitates the identification of thermal effects. The setup and the influence of radiation on the patch-clamp current are thoroughly characterized. For the independent optical verification of heating effects, a temperature microscopy technique is applied with high spatial, temporal and temperature resolution. It is shown that the effect of radiation at GHz frequencies on whole cell currents is predominantly thermal in nature in the case of RBL cells with an endogenous K(ir) 2.1 channel.     

Regulation of a Dynamic Interaction between Two Microtubule-binding Proteins,EB1 and TIP150, by the Mitotic p300/CBP-associated Factor (PCAF) Orchestrates Kinetochore Microtubule Plasticity and Chromosome Stability during Mitosis

The microtubule cytoskeleton network orchestrates cellular dynamics and chromosome stability in mitosis. Although tubulin acetylation is essential for cellular plasticity, it has remained elusive how kinetochore microtubule plus-end dynamics are regulated by p300/CBP-associated factor (PCAF) acetylation in mitosis. Here, we demonstrate that the plus-end tracking protein, TIP150, regulates dynamic kinetochore-microtubule attachments by promoting the stability of spindle microtubule plus-ends. Suppression of TIP150 by siRNA results in metaphase alignment delays and perturbations in chromosome biorientation. TIP150 is a tetramer that binds an end-binding protein (EB1) dimer through the C-terminal domains, and overexpression of the C-terminal TIP150 or disruption of the TIP150-EB1 interface by a membrane-permeable peptide perturbs chromosome segregation. Acetylation of EB1-PCAF regulates the TIP150 interaction, and persistent acetylation perturbs EB1-TIP150 interaction and accurate metaphase alignment, resulting in spindle checkpoint activation. Suppression of the mitotic checkpoint serine/threonine protein kinase, BubR1, overrides mitotic arrest induced by impaired EB1-TIP150 interaction, but cells exhibit whole chromosome aneuploidy. Thus, the results identify a mechanism by which the TIP150-EB1 interaction governs kinetochore microtubule plus-end plasticity and establish that the temporal control of the TIP150-EB1 interaction by PCAF acetylation ensures chromosome stability in mitosis.     

The Effect of TIP on Pneumovirus-Induced Pulmonary Edema in Mice

Background

Pulmonary edema plays a pivotal role in the pathophysiology of respiratory syncytial virus (RSV)-induced respiratory failure. In this study we determined whether treatment with TIP (AP301), a synthetic cyclic peptide that mimics the lectin-like domain of human TNF, decreases pulmonary edema in a mouse model of severe human RSV infection. TIP is currently undergoing clinical trials as a therapy for pulmonary permeability edema and has been shown to decrease pulmonary edema in different lung injury models.

Methods

C57BL/6 mice were infected with pneumonia virus of mice (PVM) and received TIP or saline (control group) by intratracheal instillation on day five (early administration) or day seven (late administration) after infection. In a separate set of experiments the effect of multiple dose administration of TIP versus saline was tested. Pulmonary edema was determined by the lung wet-to-dry (W/D) weight ratio and was assessed at different time-points after the administration of TIP. Secondary outcomes included clinical scores and lung cellular response.

Results

TIP did not have an effect on pulmonary edema in different dose regimens at different time points during PVM infection. In addition, TIP administration did not affect clinical severity scores or lung cellular response.

Conclusion

In this murine model of severe RSV infection TIP did not affect pulmonary edema nor course of disease.     

RVBs Are Required for Assembling a Functional TIP60 Complex

RVB1/RVB2 (RuvBL1/RuvBL2 or pontin/reptin) are enigmatic AAA⁺ ATPase proteins that are present in multiple cellular complexes. Although they have been implicated in many cellular functions, the exact molecular function of RVB proteins in the various complexes is not clear. TIP60 complex (TIP60.com) is a tumor suppressor chromatin-remodeling complex containing RVB proteins. RVBs are required for the lysine acetyltransferase activity of TIP60.com but not for that of the pure recombinant TIP60 polypeptide. Here we describe two molecular functions of RVBs in TIP60.com. First, RVBs negate the repression of catalytic activity of TIP60 by another protein in TIP60.com, p400. RVBs competitively displace the SNF2 domain of p400 from the TIP60 polypeptide. In addition RVBs are also required for heat stability of TIP60.com by a p400-independent pathway. RVB1 and RVB2 are redundant with each other for these functions and do not require their ATPase activities. Thus, RVB proteins act as molecular adaptors that can substitute for one another to facilitate the optimal assembly, heat stability, and function of the TIP60 complex.     

Hourglass SiO2 coating increases the performance of planar patch-clamp

Obtaining high-throughput electrophysiological recordings is an ongoing challenge in ion channel biophysics and drug discovery. One particular area of development is the replacement of glass pipettes with planar devices in order to increase throughput. However, successful patch-clamp recordings depend on a surface coating which ideally should promote and stabilize giga-seal formation. Here, we present data supporting the use of a structured SiO(2) coating to improve the ability of cells to form a "seal" with a planar patch-clamp substrate. The method is based on a correlation study taking into account structure and size of the pores, surface roughness and chip capacitance. The influence of these parameters on the quality of the seal was assessed. Plasma-enhanced chemical vapour deposition (PECVD) of SiO(2) led to an hourglass structure of the pore and a tighter seal than that offered by a flat, thermal SiO(2) surface. The performance of PECVD chips was validated by recording recombinant potassium channels, BK(Ca), expressed in stable HEK-293 cell lines and in inducible CHO cell lines and low conductance IRK1, and endogenous cationic currents from CHO cells. This multiparametric investigation led to the production of improved chips for planar patch-clamp applications which allow electrophysiological recordings from a wide range of cell lines.     

TIP150 interacts with and targets MCAK at the microtubule plus ends

The microtubule (MT) cytoskeleton orchestrates the cellular plasticity and dynamics that underlie morphogenesis and cell division. Growing MT plus ends have emerged as dynamic regulatory machineries in which specialized proteins—called plus-end tracking proteins (+TIPs)—bind to and control the plus-end dynamics that are essential for cell division and migration. However, the molecular mechanisms underlying the plus-end regulation by +TIPs at spindle and astral MTs have remained elusive. Here, we show that TIP150 is a new +TIP that binds to end-binding protein 1 (EB1) in vitro and co-localizes with EB1 at the MT plus ends in vivo. Suppression of EB1 eliminates the plus-end localization of TIP150. Interestingly, TIP150 also binds to mitotic centromere-associated kinesin (MCAK), an MT depolymerase that localizes to the plus end of MTs. Suppression of TIP150 diminishes the plus-end localization of MCAK. Importantly, aurora B-mediated phosphorylation disrupts the TIP150–MCAK association in vitro. We reason that TIP150 facilitates the EB1-dependent loading of MCAK onto MT plus ends and orchestrates the dynamics at the plus end of MTs.     

TIP49b, a new RuvB-like DNA helicase, is included in a complex together with another RuvB-like DNA helicase, TIP49a.

We previously reported that TIP49a is a novel mammalian DNA helicase showing structural similarity with the bacterial recombination factor RuvB. In this study, we isolated a new TIP49a-related gene, termed TIP49b, from human and yeast cells. TIP49b also resembled RuvB, thus suggesting that TIP49a and TIP49b are included in a gene family. Like TIP49a, TIP49b was abundantly expressed in the testis and thymus. Enzyme assays revealed that TIP49b was an single-stranded DNA-stimulated ATPase and ATP-dependent DNA helicase. Most of the enzymatic properties of TIP49b were the same as those of TIP49a, whereas the polarity of TIP49b DNA helicase activity (5' to 3') was the opposite to that of TIP49a. TIP49b and TIP49a bound to each other and were included in the same complex of approximately 700 kDa in a cell. We found that TIP49b was an essential gene for the growth of Saccharomyces cerevisiae, as is the TIP49a gene, suggesting that TIP49b does not complement the TIP49a function and vice versa. From these observations, we suggest that TIP49b plays an essential role in the cellular processes involved in DNA metabolism.     

Characterization of plasma triiodophenol binding proteins in vertebrates and tissue distribution of triiodophenol in Rana catesbeiana tadpoles

We investigated the interaction of 2,4,6-triiodophenol (TIP), a potent thyroid hormone disrupting chemical, with serum proteins from rainbow trout (Onchorhynchus mykiss), bullfrog (Rana catesbeiana), chicken (Gallus gallus), pig (Sus scrofa domesticus), and rat (Rattus norvegicus) using a [(125)I]TIP binding assay, gel filtration chromatography, and native polyacrylamide gel electrophoresis. [(125)I]TIP bound non-specifically to proteins in trout serum, specifically but weakly to proteins in bullfrog serum, and specifically and strongly to proteins in chicken, pig, and rat serum samples. Candidate TIP-binding proteins included lipoproteins (220-320kDa) in trout, albumin in bullfrog, albumin and transthyretin (TTR) in chicken and pig, and TTR in rat. TTR in the chicken, pig, and rat serum samples was responsible for the high-affinity, low-capacity binding sites for TIP (dissociation constant 2.2-3.5×10(-10)M). In contrast, a weak interaction of [(125)I]TIP with tadpole serum proteins accelerated [(125)I]TIP cellular uptake in vitro. Intraperitoneal injection of [(125)I]TIP in tadpoles revealed that the radioactivity was predominantly accumulated in the gallbladder and the kidney. The differences in the molecular and binding properties of TIP binding proteins among vertebrates would affect in part the cellular availability, tissue distribution and clearance of TIP.     

Recruitment of TIP47 to lipid droplets is controlled by the putative hydrophobic cleft

Adipose differentiation-related protein (ADRP) and TIP47 show sequence similarity, particularly in their N-terminal PAT-1 domain. Under standard culture conditions, ADRP existed in most lipid droplets (LDs), whereas TIP47 was observed only in some LDs and recruited to LDs on treatment with fatty acids. By analyzing deletion mutants, we found that the C-terminal half of TIP47, or more specifically the putative hydrophobic cleft [S.J. Hickenbottom, A.R. Kimmel, C. Londos, J.H. Hurley, Structure of a lipid droplet protein; the PAT family member TIP47, Structure (Camb) 12 (2004) 1199-1207.], was involved in LD targeting and responsiveness to fatty acids. The result contrasted with that observed for ADRP and implied a distinct LD-targeting mechanism for TIP47. Consistent with this, overexpression of Rab18 decreased ADRP, but not TIP47, from LDs, and TIP47 did not displace pre-existing ADRP from LDs. But ADRP may be a factor to control the TIP47 behavior, because TIP47 in LDs increased upon down-regulation of ADRP. The results suggested that the putative hydrophobic cleft is critical for the unique characteristics of TIP47.     

TIP47 is Required for the Production of Infectious HIV‐1 Particles from Primary Macrophages

Macrophages are among the major targets of HIV‐1 infection and play a key role in viral pathogenesis. Identification of the cellular cofactors involved in the production of infectious HIV‐1 from macrophages is thus crucial. Here, we investigated the role of the cellular cofactor TIP47 in HIV‐1 morphogenesis in primary macrophages. Using siRNA approach, we show that TIP47 is essential for HIV‐1 infectivity and propagation. TIP47 silencing disrupts Gag and Env colocalization in macrophages. Moreover, mutations in HIV‐1 Gag or Env, which abolish interaction with TIP47, impair HIV‐1 propagation and infectivity preventing colocalization of Gag and Env, Gag and Env coimmunoprecipitation. Interestingly, disruption of Gag‐TIP47 interaction by matrix mutation or TIP47 depletion also causes Gag to localize in scattered dots in the vicinity of the plasma membrane of macrophages. Therefore, TIP47 is required for the encounter between Gag and Env, and thus for the generation of infectious HIV‐1 particles from primary macrophages.     

TIP39 modulates effects of novelty‐induced arousal on memory

Tuberoinfundibular peptide of 39 residues (TIP39) is a neuropeptide localized to neural circuits subserving emotional processing. Recent work showed that mice with null mutation for the gene coding TIP39 (TIP39‐KO mice) display increased susceptibility to environmental provocation. Based on this stressor‐dependent phenotype, the neuroanatomical distribution of TIP39, and knowledge that novelty‐induced arousal modulates memory functions via noradrenergic activation, we hypothesized that exposure to a novel environment differently affects memory performance of mice with or without TIP39 signaling, potentially by differences in sensitivity of the noradrenergic system. We tested TIP39‐KO mice and mice with null mutation of its receptor, the parathyroid hormone 2 receptor (PTH2‐R), in tasks of short‐term declarative and social memory (object recognition and social recognition tests, respectively), and of working memory (Y‐maze test) under conditions of novelty‐induced arousal or acclimation to the test conditions. Mice lacking TIP39 signaling showed memory impairment selectively under conditions of novelty‐induced arousal. Acute administration of a PTH2‐R antagonist in wild‐type mice had a similar effect. The restoration of memory functions in TIP39‐KO mice after injection of a β‐adrenoreceptor‐blocker, propranolol, suggested involvement of the noradrenergic system. Collectively, these results suggest that the TIP39/PTH2‐R system modulates the effects of novelty exposure on memory performance, potentially by acting on noradrenergic signaling.     

TIP120A associates with cullins and modulates ubiquitin ligase activity

The cullin-containing ubiquitin-protein isopeptide ligases (E3s) play an important role in regulating the abundance of key proteins involved in cellular processes such as cell cycle and cytokine signaling. They have multisubunit modular structures in which substrate recognition and the catalysis of ubiquitination are carried out by distinct polypeptides. In a search for proteins involved in regulation of cullin-containing E3 ubiquitin ligases we immunopurified CUL4B-containing complex from HeLa cells and identified TIP120A as an associated protein by mass spectrometry. Immunoprecipitation of cullins revealed that all cullins tested specifically interacted with TIP120A. Reciprocal immunoaffinity purification of TIP120A confirmed the stable interaction of TIP120A with cullin family proteins. TIP120A formed a complex with CUL1 and Rbx1, but interfered with the binding of Skp1 and F-box proteins to CUL1. TIP120A greatly reduced the ubiquitination of phosphorylated IkappaBalpha by SCF(beta-TrCP) ubiquitin ligase. These results suggest that TIP120A functions as a negative regulator of SCF E3 ubiquitin ligases and may modulate other cullin ligases in a similar fashion.