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The neurotoxic MEC-4(d) DEG/ENaC sodium channel conducts calcium: implications for necrosis initiation

Nature Neuroscience volume 7, pages 1337–1344 (2004)Cite this article

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Abstract

Hyperactivation of the Caenorhabditis elegans MEC-4 Na+ channel of the DEG/ENaC superfamily (MEC-4(d)) induces neuronal necrosis through an increase in intracellular Ca2+ and calpain activation. How exacerbated Na+ channel activity elicits a toxic rise in cytoplasmic Ca2+, however, has remained unclear. We tested the hypothesis that MEC-4(d)-induced membrane depolarization activates voltage-gated Ca2+ channels (VGCCs) to initiate a toxic Ca2+ influx, and ruled out a critical requirement for VGCCs. Instead, we found that MEC-4(d) itself conducts Ca2+ both when heterologously expressed in Xenopus oocytes and in vivo in C. elegans touch neurons. Data generated using the Ca2+ sensor cameleon suggest that an induced release of endoplasmic reticulum (ER) Ca2+ is crucial for progression through necrosis. We propose a refined molecular model of necrosis initiation in which Ca2+ influx through the MEC-4(d) channel activates Ca2+-induced Ca2+ release from the ER to promote neuronal death, a mechanism that may apply to neurotoxicity associated with activation of the ASIC1a channel in mammalian ischemia.

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Figure 1: VGCCs do not influence mec-4(d)-induced cell death.
Figure 2: Endogenous Ca2+-activated Cl− currents are activated in Xenopus oocytes injected with mec-4(d) when exposed to CaCl2 solutions.
Figure 3: MEC-4(d) channels conduct Ca2+.
Figure 4: The MEC-4(d) subunit is required for Ca2+ permeability.
Figure 5: The Ca2+-activated Cl− current activated by exposure to CaCl2 bath is activated only in oocytes expressing functional MEC-4(d) channels.
Figure 6: Cultured mec-4(d)-expressing touch receptor neurons show an amiloride-sensitive Ca2+-current not present in mec-4(+) neurons.

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Acknowledgements

We thank W.-H. Lee and M. Lizzio for help with molecular biology; J. Pintar and M.-S. Hsu for the use of the vibrotome for cutting oocyte sections; M. Chalfie for the mec clones; and A. Galli, I. Mano and G. Patterson for critically reading the manuscript. This work was supported by grants from the National Institutes of Health (NS034435 and NS37955 to M.D., NS049511 to L.B., NSF00139 Minority Postdoctoral Fellowship to D.C.R.), from the New Jersey Commission on Spinal Cord Research, and from Psykiatrisk Forskningsfond and Novo Nordisk (to C.F.-J.) and from Fulbright and Louis Bevier Fellowships (to B.G.).

*Note: The version of this article that was published online on November 7, 2004 omitted one of the supporting National Institutes of Health grant numbers in the Acknowledgments. W.R.S. was supported by NIH grant number DA016445. The online version was corrected on 14 November 2004, and the printed version of the article is correct. This change affects the HTML and PDF versions of the article; print will be corrected before publication.

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  1. Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, A232 Nelson Biological Laboratories, 604 Allison Road, Piscataway, 08854, New Jersey, USA

    Laura Bianchi, Beate Gerstbrein, Dewey C Royal, Gargi Mukherjee, Mary Anne Royal, Jian Xue & Monica Driscoll

  2. Division of Biology, University of California, San Diego, La Jolla, 92093-0349, California, USA

    Christian Frøkjær-Jensen & William R Schafer

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Supplementary information

Supplementary Fig. 1

Properties of cultured C. elegans touch neurons. (a) Primary culture of strain bzIs18 which expresses yellow cameleon 2.12 (yc2.12) in the mechanosensory neurons. All neurons expressing cameleon show neuronal morphology. Scale bar 10 micron. (b) Example of cultured touch neuron expressing GFP under the mechanosensory neuron specific promoter pmec-4. On the bottom, the same neuron was stained with an antibody raised against acetylated α-tubulin (Sigma); a unique modification to touch neurons in vivo3. (c) C. elegans embryonic cells were cultured from pmec-4::gfp and mec-4(d);pmec-4::gfp nematodes. Cells were plated at the same density (~200,000 cells/cm2) in individual wells and the number of touch neurons/40x field (identified as expressing GFP) were counted 24 hours and 4 days after plating. Ten fields per sample were counted. (d) Experiment conducted similarly to panel c for WT and mec-4(d) touch neurons plus and minus genetic modification (calreticulin null mutant bz29) and pharmacological treatments (10 µM dantrolene or 10 µM amiloride). The number of touch neurons/40x field was determined 24 hours after plating. Ten fields per sample were counted. (JPG 25 kb)

Supplementary Fig. 2

Calibration of yellow cameleon 2.12 in cultured neurons. (a) Pseudocolor image of cultured mechanosensory neurons in low calcium (0 mM Ca2+, 5 mM EGTA) left and high calcium (10 mM Ca2+) right. Pseudocolor scale bar indicates the yellow fluorescent protein/cyan fluorescent protein (YFP/CFP) emission ratio. (b) Representative trace from cameleon calibration in cultured neurons. Prior to recording, cultures were incubated 25-30 minutes in a solution with a defined concentration of free calcium in the presence of the calcium ionophore Br-A23187 (10 µM). The solution also contained Rotenone (10mM) and 2-deoxy-D-glucose (1.8mM) to block active pumps. (c) Yc2.12 calibration curve showing the aggregate results from 4-7 cells measured at each of 11 different free calcium solutions. Ratio is normalized to the maximal ratio change for the individual cell. A biphasic calcium dependence gave the best fit, with apparent EC50 values of 0.4 µM and 40 µM. The maximum/minimum ratio change was 81 ± 2 % (n = 12). (d) Estimate of the intracellular calcium concentration in cultured neurons. Cultured neurons were imaged 3 minutes in a standard extracellular saline solution. Five mM EGTA, ionophore, Rotenone and 2-deoxy-D-glucose were added to determine the minimum ratio and 10 mM CaCl2, ionophore and metabolic blockers added to determine the maximum ratio value. Resting ratio in cultured neurons was measured to 22 ±7 % (n = 2, 18 cells) of maximal ratio change corresponding to a calcium concentration of approx. 200 nM. (JPG 47 kb)

Supplementary Fig. 3

The MEC-4(A713T/G717E) mutant channel exhibits smaller Ca2+ influx, altered sensitivity to divalent cations and altered sensitivity to amiloride, suggesting a role for G717 in divalent cation interaction and amiloride binding. (a) Average current/voltage relationships of Na+ currents generated in oocytes expressing MEC-4(d) + MEC-10(d) + MEC-2 + MEC-6 (filled squares, n=8) and MEC-4(A713T/G717E) + MEC-10(d) + MEC-2 + MEC-6 (open squares, n=6). Data were obtained from the same batch of oocytes and injected with the same amount of RNA. (b) Ca2+-activated Cl- current amplitude at -160 mV from the same oocytes shown in panel a. Data are expressed as mean ± SE and ** indicates p⩽0.01 by t Test. The MEC-4(A713T/G717E) channel shows strong inward rectification not present in the MEC-4(A713T) channel. (c) Current-voltage relationships of currents recorded from oocytes injected with mec-4(A713T/G717E)+mec-10(d)+mec-2+mec-6. Oocytes were perfused with a physiological NaCl solution (MgCl2 2mM and CaCl2 1 mM, filled squares, n=7) and with a divalent free solution (filled triangles, n=7). (d) Same as in panel a for mec-4(A713T)+mec-10(d)+mec-2+mec-6 injected oocytes (n=5). (e) Amiloride sensitivity of MEC-4(A713T/G717E). Sensitivity to 10 µM amiloride was determined at -160 using the Ca2+-activated Cl- currents as read-out of Ca2+ permeation through MEC-4 channels. The value reported for MEC-4(A713T) is obtained from figure 3g, (n=5). * designates p⩽0.05 by t Test. (JPG 26 kb)

Supplementary Table 1

Results of manipulations designed to disrupt potential sources of plasma membrane Ca+ entry on mec-4(d)-induced necrosis. (PDF 120 kb)

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Bianchi, L., Gerstbrein, B., Frøkjær-Jensen, C. et al. The neurotoxic MEC-4(d) DEG/ENaC sodium channel conducts calcium: implications for necrosis initiation. Nat Neurosci 7, 1337–1344 (2004). https://doi.org/10.1038/nn1347

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