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In JoVE (1)
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- The Journal of Biological Chemistry
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Articles by Ida Rishal in JoVE
JoVE Neuroscience
Axoplasm Isolatie van Rat heupzenuw
Department of Biological Chemistry, Weizmann Institute of Science
Demonstreren we een protocol voor axoplasm isolatie van volwassen ratten heupzenuw op basis van dissectie van de zenuw bundels en incubatie in hypotone medium om myeline release en lyseren niet-axonale structuren, gevolgd door extractie van de resterende axon-verrijkt materiaal.
Other articles by Ida Rishal on PubMed
Na+ Promotes the Dissociation Between Galpha GDP and Gbeta Gamma, Activating G Protein-gated K+ Channels
G protein-gated K(+) channels (GIRK, or Kir3) are activated by the direct binding of Gbetagamma or of cytosolic Na(+). Na(+) activation is fast, Gbetagamma-independent, and probably via a direct, low affinity (EC(50), 30-40 mm) binding of Na(+) to the channel. Here we demonstrate that an increase in intracellular Na(+) concentration, [Na(+)](in), within the physiological range (5-20 mm), activates GIRK within minutes via an additional, slow mechanism. The slow activation is observed in GIRK mutants lacking the direct Na(+) effect. It is inhibited by a Gbetagamma scavenger, hence it is Gbetagamma-dependent; but it does not require GTP. We hypothesized that Na(+) elevates the cellular concentration of free Gbetagamma by promoting the dissociation of the Galphabetagamma heterotrimer into free Galpha(GDP) and Gbetagamma. Direct biochemical measurements showed that Na(+) causes a moderate decrease (approximately 2-fold) in the affinity of interaction between Galpha(GDP) and Gbetagamma. Furthermore, in accord with the predictions of our model, slow Na(+) activation was enhanced by mild coexpression of Galpha(i3). Our findings reveal a previously unknown mechanism of regulation of G proteins and demonstrate a novel Gbetagamma-dependent regulation of GIRK by Na(+). We propose that Na(+) may act as a regulatory factor, or even a second messenger, that regulates effectors via Gbetagamma.
Mapping the Gbetagamma-binding Sites in GIRK1 and GIRK2 Subunits of the G Protein-activated K+ Channel
G protein-activated K+ channels (Kir3 or GIRK) are activated by direct binding of Gbetagamma. The binding sites of Gbetagamma in the ubiquitous GIRK1 (Kir3.1) subunit have not been unequivocally charted, and in the neuronal GIRK2 (Kir3.2) subunit the binding of Gbetagamma has not been studied. We verified and extended the map of Gbetagamma-binding sites in GIRK1 by using two approaches: direct binding of Gbetagamma to fragments of GIRK subunits (pull down), and competition of these fragments with the Galphai1 subunit for binding to Gbetagamma. We also mapped the Gbetagamma-binding sites in GIRK2. In both subunits, the N terminus binds Gbetagamma. In the C terminus, the Gbetagamma-binding sites in the two subunits are not identical; GIRK1, but not GIRK2, has a previously unrecognized Gbetagamma-interacting segments in the first half of the C terminus. The main C-terminal Gbetagamma-binding segment found in both subunits is located approximately between amino acids 320 and 409 (by GIRK1 count). Mutation of C-terminal leucines 262 or 333 in GIRK1, recognized previously as crucial for Gbetagamma regulation of the channel, and of the corresponding leucines 273 and 344 in GIRK2 dramatically altered the properties of K+ currents via GIRK1/GIRK2 channels expressed in Xenopus oocytes but did not appreciably reduce the binding of Gbetagamma to the corresponding fusion proteins, indicating that these residues are mainly important for the regulation of Gbetagamma-induced changes in channel gating rather than Gbetagamma binding.
Galphai1 and Galphai3 Differentially Interact With, and Regulate, the G Protein-activated K+ Channel
G protein-activated K(+) channels (GIRKs; Kir3) are activated by direct binding of Gbetagamma subunits released from heterotrimeric G proteins. In native tissues, only pertussis toxin-sensitive G proteins of the G(i/o) family, preferably Galpha(i3) and Galpha(i2), are donors of Gbetagamma for GIRK. How this specificity is achieved is not known. Here, using a pull-down method, we confirmed the presence of Galpha(i3-GDP) binding site in the N terminus of GIRK1 and identified novel binding sites in the N terminus of GIRK2 and in the C termini of GIRK1 and GIRK2. The non-hydrolyzable GTP analog, guanosine 5'-3-O-(thio)triphosphate, reduced the binding of Galpha(i3) by a factor of 2-4. Galpha(i1-GDP) bound to GIRK1 and GIRK2 much weaker than Galpha(i3-GDP). Titrated expression of components of signaling pathway in Xenopus oocytes and their activation by m2 muscarinic receptors revealed that G(i3) activates GIRK more efficiently than G(i1), as indicated by larger and faster agonist-evoked currents. Activation of GIRK by purified Gbetagamma in excised membrane patches was strongly augmented by coexpression of Galpha(i3) and less by Galpha(i1). Differences in physical interactions of GIRK with GDP-bound Galpha subunits, or Galphabetagamma heterotrimers, may dictate different extents of Galphabetagamma anchoring, influence the efficiency of GIRK activation by Gbetagamma, and play a role in determining signaling specificity.
Gbetagamma-dependent and Gbetagamma-independent Basal Activity of G Protein-activated K+ Channels
Cardiac and neuronal G protein-activated K+ channels (GIRK; Kir3) open following the binding of Gbetagamma subunits, released from Gi/o proteins activated by neurotransmitters. GIRKs also possess basal activity contributing to the resting potential in neurons. It appears to depend largely on free Gbetagamma, but a Gbetagamma-independent component has also been envisaged. We investigated Gbetagamma dependence of the basal GIRK activity (A(GIRK,basal)) quantitatively, by titrated expression of Gbetagamma scavengers, in Xenopus oocytes expressing GIRK1/2 channels and muscarinic m2 receptors. The widely used Gbetagamma scavenger, myristoylated C terminus of beta-adrenergic kinase (m-cbetaARK), reduced A(GIRK,basal) by 70-80% and eliminated the acetylcholine-evoked current (I(ACh)). However, we found that m-cbetaARK directly binds to GIRK, complicating the interpretation of physiological data. Among several newly constructed Gbetagamma scavengers, phosducin with an added myristoylation signal (m-phosducin) was most efficient in reducing GIRK currents. m-phosducin relocated to the membrane fraction and did not bind GIRK. Titrated expression of m-phosducin caused a reduction of A(GIRK,basal) by up to 90%. Expression of GIRK was accompanied by an increase in the level of Gbetagamma and Galpha in the plasma membrane, supporting the existence of preformed complexes of GIRK with G protein subunits. Increased expression of Gbetagamma and its constitutive association with GIRK may underlie the excessively high A(GIRK,basal) observed at high expression levels of GIRK. Only 10-15% of A(GIRK,basal) persisted upon expression of both m-phosducin and cbetaARK. These results demonstrate that a major part of Ibasal is Gbetagamma-dependent at all levels of channel expression, and only a small fraction (<10%) may be Gbetagamma-independent.
Kinetic Modeling of Na(+)-induced, Gbetagamma-dependent Activation of G Protein-gated K(+) Channels
G protein-activated K(+)(GIRK) channels are activated by numerous neurotransmitters that act on Gi/o proteins, via a direct interaction with the Gbetagamma subunit of G proteins. In addition, GIRK channels are positively regulated by intracellular Na(+) via a direct interaction (fast pathway) and via a GGbetagamma-dependent mechanism (slow pathway). The slow modulation has been proposed to arise from the recently described phenomenon of Na(+)-induced reduction of affinity of interaction between GalphaGDP and Gbetagamma subunits of G proteins. In this scenario, elevated Na(+) enhances basal dissociation of G protein heterotrimers, elevating free cellular Gbetagamma and activating GIRK. However, it is not clear whether this hypothesis can account for the quantitative and kinetic aspects of the observed regulation. Here, we report the development of a quantitative model of slow, Na(+)-dependent, G protein-mediated activation of GIRK. Activity of GIRK1F137S channels, which are devoid of direct interaction with Na(+), was measured in excised membrane patches and used as an indicator of free GGbetagamma levels. The change in channel activity was used to calculate the Na(+)-dependent change in the affinity of G protein subunit interaction. Under a wide range of initial conditions, the model predicted that a relatively small decrease in the affinity of interaction of GalphaGDP and GGbetagamma (about twofold under most conditions) accounts for the twofold activation of GIRK induced by Na(+), in agreement with biochemical data published previously. The model also correctly described the slow time course of Na(+) effect and explained the previously observed enhancement of Na(+)-induced activation of GIRK by coexpressed Galphai3. This is the first quantitative model that describes the basal equilibrium between free and bound G protein subunits and its consequences on regulation of a GGbetagamma effector.
Amplitude Histogram-based Method of Analysis of Patch Clamp Recordings That Involve Extreme Changes in Channel Activity Levels
Many ion channels show low basal activity, which is increased hundreds-fold by the relevant gating factor. A classical example is the activation G-protein-activated K(+) channels (GIRK) by Gbetagamma subunit dimer. The extent of activation (relative to basal current), R(a), is an important physiological parameter, usually readily estimated from whole cell recordings. However, calculation of R(a) often becomes non-trivial in multi-channel patches because of extreme changes in activity upon activation, from a seemingly single-channel pattern to a macroscopic one. In such cases, calculation of the net current flowing through the channels in the patch, I, before and after activation may require different methods of analysis. To address this problem, we utilized neuronal GIRK channels activated by purified Gbetagamma in excised patches of Xenopus oocytes. Channels were expressed at varying densities, from a few to several hundreds per patch. We present a simple and fast method of calculating I using amplitude histogram analysis and establish its accuracy by comparing with I calculated from event lists. This method allows the analysis of extreme changes in I in multichannel patches, which would be impossible using the standard methods of idealization and event list generation.
Retrograde Signaling in Axonal Regeneration
Neuronal regeneration in the peripheral nervous system requires mobilization of intrinsic neurite outgrowth mechanisms. This process depends on retrograde signaling between lesion site and soma to provide accurate and timely information on the nature and extent of axonal damage, and to elicit an appropriate cell body response. An early phase of electrophysiological signaling is followed by an ensemble of motor-driven signals, some of which are dependent on local protein translation in the axon and formation of an importins-coordinated retrograde complex. In addition to eliciting the cell body response, computational analyses suggest that this biphasic mechanism may provide information on the distance of the leson site from the neuronal cell body. Encouraging recent data suggest that it may be possible to apply this emerging understanding of retrograde signaling mechanisms to activate intrinsic regeneration mechanisms also in growth-refractory central neurons.
Axoplasm Isolation from Peripheral Nerve
Localized changes in the composition of axonal cytoplasm (axoplasm) are critical for many biological processes, including axon guidance, responses to injury, neurite outgrowth, and axon-glia interactions. Biochemical and molecular studies of these mechanisms have been heavily focused on in vitro systems because of the difficulty of obtaining subcellular extracts from mammalian tissues in vivo. As in vitro systems might not replicate the in vivo situation, reliable methods of axoplasm extraction from whole nerve would be helpful for mechanistic studies on axons. Here we develop and evaluate a new procedure for preparation of axoplasm from rat peripheral nerve, based on incubation of separated short segements of nerve fascicles in hypotonic medium to separate myelin and lyse nonaxonal structures, followed by extraction of the remaining axon-enriched material. We show that this new procedure reduces serum and glial cell contamination and facilitates proteomic analyses of axonal contents.
Signaling to Transcription Networks in the Neuronal Retrograde Injury Response
Retrograde signaling from axon to soma activates intrinsic regeneration mechanisms in lesioned peripheral sensory neurons; however, the links between axonal injury signaling and the cell body response are not well understood. Here, we used phosphoproteomics and microarrays to implicate approximately 900 phosphoproteins in retrograde injury signaling in rat sciatic nerve axons in vivo and approximately 4500 transcripts in the in vivo response to injury in the dorsal root ganglia. Computational analyses of these data sets identified approximately 400 redundant axonal signaling networks connected to 39 transcription factors implicated in the sensory neuron response to axonal injury. Experimental perturbation of individual overrepresented signaling hub proteins, including Abl, AKT, p38, and protein kinase C, affected neurite outgrowth in sensory neurons. Paradoxically, however, combined perturbation of Abl together with other hub proteins had a reduced effect relative to perturbation of individual proteins. Our data indicate that nerve injury responses are controlled by multiple regulatory components, and suggest that network redundancies provide robustness to the injury response.
Behavioral and Other Phenotypes in a Cytoplasmic Dynein Light Intermediate Chain 1 Mutant Mouse
The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system.
Axonal Transcription Factors Signal Retrogradely in Lesioned Peripheral Nerve
Retrograde axonal injury signalling stimulates cell body responses in lesioned peripheral neurons. The involvement of importins in retrograde transport suggests that transcription factors (TFs) might be directly involved in axonal injury signalling. Here, we show that multiple TFs are found in axons and associate with dynein in axoplasm from injured nerve. Biochemical and functional validation for one TF family establishes that axonal STAT3 is locally translated and activated upon injury, and is transported retrogradely with dynein and importin α5 to modulate survival of peripheral sensory neurons after injury. Hence, retrograde transport of TFs from axonal lesion sites provides a direct link between axon and nucleus.
