Table of Contents

Volume 4, Issue 5

  September/October 2010

  Pages 335 - 428

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COMMENTARY

Open Access Article

Channel triage: Emerging insights into the processing and quality control of hERG potassium channels by DnaJA proteins 1, 2 and 4

Eva Ahrendt and Janice E.A. Braun
Pages 335 - 336
http://dx.doi.org/10.4161/chan.4.5.13090
PDF




ARTICLE ADDENDA

Open Access Article

Targeting K_V channels rescues retinal ganglion cells in vivo directly and by reducing inflammation

Paulo Koeberle and Lyanne C. Schlichter
Pages 337 - 346
http://dx.doi.org/10.4161/chan.4.5.12790
Abstract | PDF

Retinal ganglion cell (RGC) degeneration is an important cause of visual impairment, and results in part from microglia-mediated inflammation. Numerous experimental studies have focused on identifying drug targets to rescue these neurons. We recently showed that K_V1.1 and K_V1.3 channels are expressed in adult rat RGCs and that siRNA-mediated knockdown of either channel reduces RGC death after optic nerve transection. Earlier we found that K_V1.3 channels also contribute to microglial activation and neurotoxicity; raising the possibility that these channels contribute to neurodegeneration through direct roles in RGCs and through inflammatory mechanisms. Here, RGC survival was increased by combined siRNA-mediated knockdown of K_V1.1 and K_V1.3 in RGCs, but survival was much greater when knockdown of either channel was combined with intraocular injection of a K_V1.3 channel blocker (agitoxin-2 or margatoxin). After axotomy, increased expression of several inflammation-related molecules preceded RGC loss and, consistent with a dual mechanism, their expression was differentially affected when channel knockdown in RGCs was combined with K_V1.3 blocker injection. K_V1.3 blockers reduced activation of retinal microglia and their tight apposition along RGC axon fascicles after axotomy, but did not prevent their migration from the inner plexiform to the damaged ganglion cell layer. Expression of several growth factors increased after axotomy; and again, there were differences following blocker injection compared with RGC-selective channel knockdown. These results provide evidence that K_V1.3 channels play important roles in apoptotic degeneration of adult RGCs through cell-autonomous mechanisms mediated by channels in the neurons, and non-autonomous mechanisms mediated by microglia and inflammation.

Open Access Article

The role of STEP in Alzheimer's disease

Pradeep Kurup, Yongfang Zhang, Deepa V. Venkitaramani, Jian Xu and Paul J. Lombroso
Pages 347 - 350
http://dx.doi.org/10.4161/chan.4.5.12910
Abstract | PDF

Amyloid beta (Aβ), the putative causative agent in Alzheimer's disease, is known to affect glutamate receptor trafficking. Previous studies have shown that Aβ down-regulates the surface expression of N-methyl D-aspartate type glutamate receptors (NMDARs) by the activation of STriatal-Enriched protein tyrosine Phosphatase 61 (STEP₆₁). More recent findings confirm that STEP₆₁ plays an important role in Aβ-induced NMDAR endocytosis. STEP levels are elevated in human AD prefrontal cortex and in the cortex of several AD mouse models. The increase in STEP₆₁ levels and activity contribute to the removal of GluN1/GluN2B receptor complexes from the neuronal surface membranes. The elevation of STEP₆₁ is due to disruption in the normal degradation of STEP₆₁ by the ubiquitin proteasome system. Here, we briefly discuss additional studies in support of our hypothesis that STEP₆₁ contributes to aspects of the pathophysiology in Alzheimer's disease. Exogenous application of Aβ-enriched conditioned medium (7PA2-CM) to wild-type cortical cultures results in a loss of GluN1/GluN2B subunits from neuronal membranes. Aβ-mediated NMDAR internalization does not occur in STEP knock-out cultures, but is rescued by the addition of active TAT-STEP to the cultures prior to Aβ treatment.

Open Access Article

Further characterization of regulation of Ca_V2.2 by Stargazin

Isabella Tselnicker and Nathan Dascal
Pages 351 - 354
http://dx.doi.org/10.4161/chan.4.5.13504
Abstract | PDF

Stargazin, a transmembrane protein expressed in the nervous system, shares similarities with the γ1 subunit of skeletal muscle calcium channels. It was thus termed γ2 subunit of neuronal calcium channels. Stargazin downregulates the expression of Ca_V2 channels, however, its functional modulation of these channels remains debated. We have reported that Stargazin modulates Ca_V2.2 channel by a Gβγ-dependent mechanism and suggested that Stargazin is not a true subunit of this channel, since all its effects on channel function are dependent on the presence of Gβγ. Moreover, Stargazin also modulated the GIRK channel in a Gβγ-dependent fashion. Here we report that Gβγ-dependent modulation by Stargazin of the biophysical properties of Ca_V2.2 is unrelated to its negative effect on channel expression and current amplitude. Finally, we suggest that this Gβγ dependent modulation of Stargazin may have physiological relevance, since it was still present when we used Ca²⁺ as charge carrier, instead of Ba²⁺




RESEARCH PAPERS

Open Access Article

Evolutionary coupling in the K_V1.2-β₂ complex

Shreedhar Natarajan, Robert J. Mashl and Eric Jakobsson
Pages 355 - 374
http://dx.doi.org/10.4161/chan.4.5.12813
Abstract | PDF

K_V1.2 is a potassium channel protein whose electrophysiological properties, including an oxygen sensing response, are modulated by auxiliary beta subunits. The beta subunits are homologous to oxidoreductases, supporting a hypothesis that the coupling of the two subunits helps connect the redox state of the cell to the electrical activity of the membrane. However the exact mechanism of the coupling has not been discovered to-date.
We apply evolutionary correlation analysis to infer previously unknown components of the interaction network regulating the response to hypoxia of the K_V1.2/β₂ complex. Briefly, evolutionary correlation analysis involves finding correlated amino acid substitutions in functionally equivalent proteins (for both subunits) across a range of species. The method thus depends on a reliable method of inferring functionally equivalent (orthologous) proteins in different species, which method we describe in a paper recently published in PLoS ONE. One key finding is the characterization of a network of motif interactions between the α and the β subunits. By significance testing, we show that the likelihood of the correlations shown in the K_V1.2-β₂ two interaction motifs arising by chance is less than 0.0003, which shows that the correlations are statistically highly significant. We therefore believe that the correlations are likely to be biologically relevant. Other major findings are correlations between specific motifs in the K_V1.2-β₂ complex and motifs in other proteins such as RACK1 and Eif36sip, which in turn are connected to the hypoxia response. Our paper combines this correlation with the literature evidence on potassium channel inactivation and hypoxia response to identify specific motifs to serve as experimental targets for studies focused on this response.
This work aims to add to our general understanding of two major issues in ion channel science: 1) how multi-protein complexes including ion channels function in coordinated fashion, and 2) how ion channels mediate the conversation between the intracellular and extracellular environments. We also aim to apply to ion channel science the principle that domain-domain interactions in proteins can be inferred from correlation of amino-acid substitutions in sets of functionally equivalent proteins.

Open Access Article

Splice-variant changes of the Ca_V3.2 T-type calcium channel mediate voltage-dependent facilitation and associate with cardiac hypertrophy and development

Laurence S. David, Esperanza Garcia, Stuart M. Cain, Elana Thau, John R. Tyson and Terrance P. Snutch
Pages 375 - 389
http://dx.doi.org/10.4161/chan.4.5.12874
Abstract | PDF

Low voltage-activated T-type calcium (Ca) channels contribute to the normal development of the heart and are also implicated in pathophysiological states such as cardiac hypertrophy. Functionally distinct T-type Ca channel isoforms can be generated by alternative splicing from each of three different T-type genes (Ca_V3.1, Ca_V3.2,Ca_V3 .3), although it remains to be described whether specific splice variants are associated with developmental states and pathological conditions. We aimed to identify and functionally characterize Ca_V3.2 T-type Ca channel alternatively spliced variants from newborn animals and to compare with adult normotensive and spontaneously hypertensive rats (SHR). DNA sequence analysis of full-length Ca_V3.2 cDNA generated from newborn heart tissue identified ten major regions of alternative splicing, the more common variants of which were analyzed by quantitative real-time PCR (qRT-PCR) and also subject to functional examination by whole-cell patch clamp. The main findings are that: (1) cardiac Ca_V3.2 T-type Ca channels are subject to considerable alternative splicing, (2) there is preferential expression ofCa_V3 .2(-25) splice variant channels in newborn rat heart with a developmental shift in adult heart that results in approximately equal levels of expression of both (+25) and (-25) exon variants, (3) in the adult stage of hypertensive rats there is a both an increase in overallCa_V3 .2 expression and a shift towards expression of Ca_V3.2(+25) containing channels as the predominant form, and (4) alternative splicing confers a variant-specific voltage-dependent facilitation ofCa_V3 .2 channels. We conclude that Ca_V3.2 alternative splicing generates significant T-type Ca channel structural and functional diversity with potential implications relevant to cardiac developmental and pathophysiological states.

Open Access Article

Random mutagenesis screening indicates the absence of a separate H⁺-sensor in the pH-sensitive Kir channels

Jennifer J. Paynter, Lijun Shang, Murali K. Bollepalli, Thomas Baukrowitz and Stephen J. Tucker
Pages 390 - 397
http://dx.doi.org/10.4161/chan.4.5.13006
Abstract | PDF

Several inwardly-rectifying (Kir) potassium channels (Kir1.1, Kir4.1 and Kir4.2) are characterised by their sensitivity to inhibition by intracellular H⁺ within the physiological range. The mechanism by which these channels are regulated by intracellular pH has been the subject of intense scrutiny for over a decade, yet the molecular identity of the titratable pH-sensor remains elusive. In this study we have taken advantage of the acidic intracellular environment of S. cerevisiae and used a K⁺-auxotrophic strain to screen for mutants of Kir1.1 with impaired pH-sensitivity. In addition to the previously identified K80M mutation, this unbiased screening approach identified a novel mutation (S172T) in the second transmembrane domain (TM2) that also produces a marked reduction in pH-sensitivity through destabilization of the closed-state. However, despite this extensive mutagenic approach, no mutations could be identified which removed channel pH-sensitivity or which were likely to act as a separate H⁺-sensor unique to the pH-sensitive Kir channels. In order to explain these results we propose a model in which the pH-sensing mechanism is part of an intrinsic gating mechanism common to all Kir channels, not just the pH-sensitive Kir channels. In this model, mutations which disrupt this pH-sensor would result in an increase, not reduction, in pH-sensitivity. This has major implications for any future studies of Kir channel pH-sensitivity and explains why formal identification of these pH-sensing residues still represents a major challenge.

Open Access Article

The N-terminal domain of Orai3 determines selectivity for activation of the store-independent ARC channel by arachidonic acid

Jill Thompson, Olivier Mignen and Trevor J. Shuttleworth
Pages 398 - 410
http://dx.doi.org/10.4161/chan.4.5.13226
Abstract | PDF

Although highly selective Ca²⁺ entry pathways play a critical role in agonist-activated Ca²⁺ signals in non-excitable cells, only with the recent discovery of the Orai proteins have the first insights into the molecular nature of these pathways been possible. To date, just two such highly Ca²⁺-selective "Orai channels" have been identified in native cells - the store-operated CRAC channels and the store-independent, arachidonic acid-activated ARC channels. Studies have shown that the functional CRAC channel pore is formed by a tetrameric arrangement of Orai1 subunits, whilst a heteropentamer of three Orai1 subunits and two Orai3 subunits forms the functional ARC channel pore. Importantly, this inclusion of Orai3 subunits in the ARC channel structure has been shown to play a specific role in determining the selectivity of these channels for activation by arachidonic acid. Using an approach based on the expression of various concatenated constructs, we examined the basis for this Orai3-dependent effect on selectivity for arachidonic acid. We show that, whilst heteropentamers containing only one Orai3 subunit are sensitive to arachidonic acid, specific selectivity for activation by this fatty acid is only achieved on inclusion of the second Orai3 subunit in the pentamer. Further studies identified the cytosolic N-terminal domain of Orai3 as the region specifically responsible for this switch in selectivity. Substitution of just this domain into an otherwise complete single Orai1 subunit within a concatenated 31111 pentamer is sufficient to change the resulting channel from one that is predominantly store-operated, to one that is exclusively activated by arachidonic acid.




REVIEW

Open Access Article

The role of G proteins in assembly and function of Kir3 inwardly rectifying potassium channels

Peter Zylbergold, Nitya Ramakrishnan and Terry Hébert
Pages 411 - 421
http://dx.doi.org/10.4161/chan.4.5.13327
Abstract | PDF

Kir3 channels (also known as GIRK channels) are important regulators of electrical excitability in both cardiomyocytes and neurons. Much is known regarding the assembly and function of these channels and the roles that their interacting proteins play in controlling these events. Further, they are one of the best studied effectors of heterotrimeric G proteins in general and Gβγ subunits in particular. However, our understanding of the roles of multiple Gβγ binding sites on Kir3 channels is still rudimentary. We discuss potential roles for Gβγ in channel assembly and trafficking in addition to their known role in cellular signaling.




TECHNICAL REPORT

Open Access Article

Simulating complex ion channel kinetics with IonChannelLab

Jose A. De Santiago-Castillo, Manuel Covarrubias, Jorge E. Sánchez-Rodríguez, Patricia Perez-Cornejo and Jorge Arreola
Pages 422 - 428
http://dx.doi.org/10.4161/chan.4.5.13404
Abstract | PDF

In silico simulation based on Markov chains is a powerful way to describe and predict the activity of many transport proteins including ion channels. However, modeling and simulation using realistic models of voltage- or ligand-gated ion channels exposed to a wide range of experimental conditions require building complex kinetic schemes and solving complicated differential equations. To circumvent these problems, we developed IonChannelLab a software tool that includes a user-friendly Graphical User Interface and a simulation library. This program supports channels with Ohmic or Goldman-Hodgkin-Katz behavior and can simulate the time-course of ionic and gating currents, single channel behavior and steady-state conditions. The program allows the simulation of experiments where voltage, ligand and ionic concentration are varied independently or simultaneously.