Molecular characterization of a swelling-induced chloride conductance regulatory protein, plCIn

doi:10.1016/0092-8674(94)90109-0

Cell

Volume 76, Issue 3, 11 February 1994, Pages 439-448

https://doi.org/10.1016/0092-8674(94)90109-0 Get rights and content

Abstract

Cells maintain control of their volume by the passage of KCI and water across their membranes, but the regulatory proteins are unknown. Expression in Xenopus oocytes of a novel protein, pl_Cln, activated a chloride conductance. We have cloned analogs of pl_Cln from rat heart and Xenopus ovary. pl_Cln was identified as an abundant soluble cytosolic protein (∼40 kd) that does not immunolocalize with the plasma membrane. pl_Cln was found in epithelial and cardiac cells, brain, and Xenopus oocytes, forming complexes with soluble actin and other cytosolic proteins. Monoclonal antibodies recognizing pl_Cln blocked activation of a native hypotonicity-induced chloride conductance (I_Cl.swell) in Xenopus oocytes, suggesting that pl_Cln may link actin-bound cytoskeletal elements to an unidentified volume-sensitive chloride channel. The high degree of sequence conservation and widespread expression of pl_Cln suggest that it is an important element in cellular volume regulation.

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Citation Excerpt :
Furthermore, pICln was later shown to be a spliceosome component (Pu, Krapivinsky, Krapivinsky, & Clapham, 1999). On the other hand, pICln was found to modulate VSOR activity in a number of cell types (Chen, Wang, & Jacob, 1999; Gschwentner et al., 1995; Hubert et al., 2000; Krapivinsly et al., 1994). pICln is known to form complexes with soluble actin and other cytosolic proteins (Krapivinsky, Ackerman, Gordon, Krapivinsky, & Clapham, 1994) and also interact with a homolog of the p21-activated protein kinases (PAKs) which is involved in cytoskeletal rearrangements (Krapivinsky, Pu, Wickman, Krapivinsky, & Clapham, 1998).
An elaborate volume regulation system based on interplay of ion channels and transporters was evolved to cope with constant osmotic challenges caused by intensive metabolism, transport and other physiological/pathophysiological events. In animal cells, two types of anion channels are directly activated by cell swelling and involved in the regulatory volume decrease (RVD): volume-sensitive outwardly rectifying anion channel (VSOR), also called volume-regulated anion channel (VRAC), and Maxi-Cl which is the most major type of maxi-anion channel (MAC). These two channels have very different biophysical profiles and exhibit opposite dependence on intracellular ATP. After several decades of verifying many false-positive candidates for VSOR and Maxi-Cl, LRRC8 family proteins emerged as major VSOR components, and SLCO2A1 protein as a core of Maxi-Cl. Still, neither of these proteins alone can fully reproduce the native channel phenotypes suggesting existence of missing components. Although both VSOR and Maxi-Cl have pores wide enough to accommodate bulky ATP⁴⁻ and MgATP²⁻ anions, evidence accumulated hitherto, based on pharmacological and gene silencing experiments, suggests that Maxi-Cl, but not VSOR, serves as one of the major pathways for the release of ATP from swollen and ischemic/hypoxic cells. Relations of VSOR and Maxi-Cl with diseases and their selective pharmacology are the topics promoted by recent advance in molecular identification of the two volume-activated, volume-regulatory anion channels.
The Function of Survival Motor Neuron Complex and Its Role in Spinal Muscular Atrophy Pathogenesis
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The survival motor neuron (SMN) protein is the centerfold of a large macromolecular complex comprising SMN, Gemins 2–8, and Unrip. The complex’s best characterized function is in the assembly of a protein ring (Sm core) on small nuclear RNAs (snRNAs), thereby forming small nuclear ribonucleoproteins (snRNPs), the major subunits of cells’ protein-coding mRNA-splicing machine. Biochemical studies identified SMN complex subunits and dissected how this first-of-its-kind RNP assembly device operates an snRNP biogenesis pathway. Atomic structures of a key intermediate provide important mechanistic insights and explain the basis of several spinal muscular atrophy (SMA)-causing SMN mutations. The SMN complex’s interactions with multiple RNA-binding proteins suggest diverse additional roles in RNA metabolism, consistent with SMA’s widespread transcriptome perturbations. Further research on the SMN complex is motivated by its central role in biology and in SMA pathogenesis, aiming to advance prospects of therapy for SMA and potentially other diseases.
Identification of Methylosome Components as Negative Regulators of Plant Immunity Using Chemical Genetics
2016, Molecular Plant
Citation Excerpt :
pICln may also play a role in maintaining cellular osmotic homeostasis during cell swelling by inducing chloride conductance (Sardini et al., 2003). Interestingly, pICln was not detected on the plasma membrane in MDCK cells using immunofluorescent staining (Krapivinsky et al., 1994), suggesting pICln to be a channel regulator rather than a channel itself. The anchor-insertion channel hypothesis may help explain these seeming controversial observations that pICln is mainly cytosolic but might be inserted into the plasma membrane under certain conditions (Strange, 1998).
Nucleotide-binding leucine-rich repeat (NLR) proteins serve as immune receptors in both plants and animals. To identify components required for NLR-mediated immunity, we designed and carried out a chemical genetics screen to search for small molecules that can alter immune responses in Arabidopsis thaliana. From 13 600 compounds, we identified Ro 8-4304 that was able to specifically suppress the severe autoimmune phenotypes of chs3-2D (chilling sensitive 3, 2D), including the arrested growth morphology and heightened PR (Pathogenesis Related) gene expression. Further, six Ro 8-4304 insensitive mutants were uncovered from the Ro 8-4304-insensitive mutant (rim) screen using a mutagenized chs3-2D population. Positional cloning revealed that rim1 encodes an allele of AtICln (I, currents; Cl, chloride; n, nucleotide). Genetic and biochemical analysis demonstrated that AtICln is in the same protein complex with the methylosome components small nuclear ribonucleoprotein D3b (SmD3b) and protein arginine methyltransferase 5 (PRMT5), which are required for the biogenesis of small nuclear ribonucleoproteins (snRNPs) involved in mRNA splicing. Double mutant analysis revealed that SmD3b is also involved in the sensitivity to Ro 8-4304, and the prmt5-1 chs3-2D double mutant is lethal. Loss of AtICln, SmD3b, or PRMT5 function results in enhanced disease resistance against the virulent oomycete pathogen Hyaloperonospora arabidopsidis Noco2, suggesting that mRNA splicing plays a previously unknown negative role in plant immunity. The successful implementation of a high-throughput chemical genetic screen and the identification of a small-molecule compound affecting plant immunity indicate that chemical genetics is a powerful tool to study whole-organism plant defense pathways.
The molecular and functional interaction between ICln and HSPC038 proteins modulates the regulation of cell volume
2011, Journal of Biological Chemistry
Identifying functional partners for protein/protein interactions can be a difficult challenge. We proposed the use of the operon structure of the Caenorhabditis elegans genome as a “new gene-finding tool” (Eichmüller, S., Vezzoli, V., Bazzini, C., Ritter, M., Fürst, J., Jakab, M., Ravasio, A., Chwatal, S., Dossena, S., Bottà, G., Meyer, G., Maier, B., Valenti, G., Lang, F., and Paulmichl, M. (2004) J. Biol. Chem. 279, 7136–7146) that could be functionally translated to the human system. Here we show the validity of this approach by studying the predicted functional interaction between ICln and HSPC038. In C. elegans, the gene encoding for the ICln homolog (icln-1) is embedded in an operon with two other genes, Nx (the human homolog of Nx is HSPC038) and Ny. ICln is a highly conserved, ubiquitously expressed multifunctional protein that plays a critical role in the regulatory volume decrease after cell swelling. Following hypotonic stress, ICln translocates from the cytosol to the plasma membrane, where it has been proposed to participate in the activation of the swelling-induced chloride current (ICl_swell). Here we show that the interaction between human ICln and HSPC038 plays a role in volume regulation after cell swelling and that HSPC038 acts as an escort, directing ICln to the cell membrane after cell swelling and facilitating the activation of ICl_swell. Assessment of the NMR structure of HSPC038 showed the presence of a zinc finger motif. Moreover, NMR and additional biochemical techniques enabled us to identify the putative ICln/HSPC038 interacting sites, thereby explaining the functional interaction of both proteins on a molecular level.
Role of pICLn in methylation of Sm proteins by PRMT5
2009, Journal of Biological Chemistry
pICln is an essential, highly conserved 26-kDa protein whose functions include binding to Sm proteins in the cytoplasm of human cells and mediating the ordered and regulated assembly of the cell’s RNA-splicing machinery by the survival motor neurons complex. pICln also interacts with PRMT5, the enzyme responsible for generating symmetric dimethylarginine modifications on the carboxyl-terminal regions of three of the canonical Sm proteins. To better understand the role of pICln in these cellular processes, we have investigated the properties of pICln and pICln·Sm complexes and the effects that pICln has on the methyltransferase activity of PRMT5. We find that pICln is a monomer in solution, binds with high affinity (K_d ∼ 160 nm) to SmD3-SmB, and forms 1:1 complexes with Sm proteins and Sm protein subcomplexes. The data support an end-capping model of pICln binding that supports current views of how pICln prevents Sm oligomerization on illicit RNA substrates. We have found that by co-expression with pICln, recombinant PRMT5 can be produced in a soluble, active form. PRMT5 alone has promiscuous activity toward a variety of known substrates. In the presence of pICln, however, PRMT5 methylation of Sm proteins is stimulated, but methylation of histones is inhibited. We have also found that mutations in pICln that do not affect Sm protein binding can still have a profound effect on the methyltransferase activity of the PRMT5 complex. Together, the data provide insights into pICln function and represent an important starting point for biochemical analyses of PRMT5.
Anion Channels
2008, Seldin and Giebisch's The Kidney

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^★: The first two authors contributed equally to this work.

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Cell

ArticleMolecular characterization of a swelling-induced chloride conductance regulatory protein, plCIn

Abstract

Meth. Enzymol.

Cell

Cell

Cell

Curr. Topics Membr. Trans.

Biochim. Biophys. Acta

Gene

Hypotonicity activates a native chloride current in Xenopus oocytes

J. Gen. Physiol.

Anisosmotic cell volume regulation: a comparative view

Am. J. Physiol.

Alternate pathways for chloride conductance activation in normal and cystic fibrosis airway epithelial cells

Am. J. Physiol.

Human Sos1: a guanine nucleotide exchange factor for ras that binds to GRB2

Science

Translation of eukaryotic messenger RNA in Xenopus oocytes

Volume-sensitive chloride conductance in bovine chromaffin cell membrane

J. Physiol.

Association of sos ras exchange protein with GRB2 is implicated in tyrosine kinase signal transduction and transformation

Nature

Involvement of calcium and cytoskeleton in gallbladder epithelial cell volume regulation

Am. J. Physiol.

Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume

Nature

Membrane mechanisms in volume and pH regulation in vertebrate cells

Physiol. Rev.

Swelling-activated KCl co-transport in rabbit red cells: flux is determined mainly by cell volume rather than shape

Am. J. Physiol.

Volume-regulatory CI-channel currents in cultured human epithelial cells

J. Physiol.

Properties and regulation of chloride channels in cystic fibrosis and normal airway cells

Pflügers Arch.

Article
Molecular characterization of a swelling-induced chloride conductance regulatory protein, pl_CIn