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The role of ion channels and intracellular metal ions in apoptosis of Xenopus oocytes
Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Apoptosis is one type of programmed cell death, important during tissue development and to maintain the tissue homeostasis. Apoptosis comprises a complex network of internal signaling pathways, and an important part of this signaling network is the action of voltage‐gated ion channels. The aim of this thesis was to explore the role of ion channels and the role of intracellular metal ions during apoptosis in Xenopus laevis oocytes. The reasons for using these oocytes are that they are large, robust, easy to handle, and easy to study electrophysiologically. Apoptosis was induced either chemically by incubation of the oocytes in staurosporine (STS) or mechanically by centrifugation of the oocytes. Ion currents were measured by a two‐electrode voltage clamp technique, intracellular ion concentrations were measured either directly by in‐house developed K+‐selective microelectrodes or indirectly by the electrophysiological technique, and apoptosis was measured by caspase‐3 activation. Paper I describes that the intracellular K+ concentration was reduced by about 30 % during STS‐induced apoptosis. However, this reduction was prevented by excessive expression of exogenous ion channels. Despite the magnitude of the intracellular K+ concentration, either normal or reduced level, the oocytes displayed normal signs of apoptosis, suggesting that the intracellular K+ reduction was not required for the apoptotic process. Because the intracellular K+ concentration was not critical for apoptosis we searched for other ion fluxes by exploring the electrophysiological properties of X. laevis oocytes. Paper II, describes a non‐inactivating Na+ current activated at positive membrane voltages that was upregulated by a factor of five during STS‐induced apoptosis. By preventing influx of Na+, the apoptotic signaling network involving capsase‐3 was prevented. To molecularly identify this voltage‐gated Na channel, the X. tropicalis genome and conserved regions of the human SCNA genes were used as a map. Paper III, shows that the voltage‐gated Na channel corresponds to the SCN2A gene ortholog and that supression of this SCN2A ortholog using miRNA prevented cell death. In conclusion, this thesis work demonstrated that a voltage‐gated Na channel is critical for the apoptotic process in X. laevis oocytes by increasing the intracellular Na+ concentration.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2014. , 47 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1424
National Category
Clinical Medicine Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-111045DOI: 10.3384/diss.diva-111045ISBN: 9789175192208 (print)OAI: oai:DiVA.org:liu-111045DiVA: diva2:752792
Public defence
2014-11-07, Berzeliussalen, Campus US, Linköpings universitet, Linköping, 13:15 (English)
Opponent
Supervisors
Available from: 2014-10-06 Created: 2014-10-06 Last updated: 2017-12-15Bibliographically approved
List of papers
1. Intracellular potassium (K+) concentration decrease is not obligatory for apoptosis
Open this publication in new window or tab >>Intracellular potassium (K+) concentration decrease is not obligatory for apoptosis
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2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 46, 39823-39828 p.Article in journal (Refereed) Published
Abstract [en]

K+ efflux is observed as an early event in the apoptotic process in various cell types. Loss of intracellular K+ and subsequent reduction in ionic strength is suggested to release the inhibition of proapoptotic caspases. In this work, a new K+-specific microelectrode was used to study possible alterations in intracellular K+ in Xenopus laevis oocytes during chemically induced apoptosis. The accuracy of the microelectrode to detect changes in intracellular K+ was verified with parallel electrophysiological measurements within the same cells. In concordance with previous studies on other cell types, apoptotic stimuli reduced the intracellular K+ concentration in Xenopus oocytes and increased caspase-3 activity. The reduction in intracellular K+ was prevented by dense expression of voltage-gated K (Kv) channels. Despite this, the caspase-3 activity was increased similarly in Kv channel expressing oocytes as in oocytes not expressing Kv channels. Thus, in Xenopus oocytes caspase-3 activity is not dependent on the intracellular concentration of K+.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2011
Keyword
Caspase-3 activation, Electrophysiology, Intracellular K+ concentrations, K+-selective microelectrode, Xenopus laevis oocytes
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-68853 (URN)10.1074/jbc.M111.262725 (DOI)000296925700016 ()
Note
Funding agencies|Swedish Research Council||Swedish Heart-Lung Foundation||Swedish Brain Foundation||County Council of Ostergotland, King Gustaf V and Queen Victorias Freemasons Foundation||Swedish Society for Medical Research||Available from: 2011-06-08 Created: 2011-06-08 Last updated: 2017-12-11Bibliographically approved
2. A Voltage Dependent Non-Inactivating Na+ Channel Activated during Apoptosis in Xenopus Oocytes
Open this publication in new window or tab >>A Voltage Dependent Non-Inactivating Na+ Channel Activated during Apoptosis in Xenopus Oocytes
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 2, 0088381- p.Article in journal (Refereed) Published
Abstract [en]

Ion channels in the plasma membrane are important for the apoptotic process. Different types of voltage-gated ion channels are up-regulated early in the apoptotic process and block of these channels prevents or delays apoptosis. In the present investigation we examined whether ion channels are up-regulated in oocytes from the frog Xenopus laevis during apoptosis. The two-electrode voltage-clamp technique was used to record endogenous ion currents in the oocytes. During staurosporine-induced apoptosis a voltage-dependent Na+ current increased three-fold. This current was activated at voltages more positive than 0 mV (midpoint of the open-probability curve was +55 mV) and showed almost no sign of inactivation during a 1-s pulse. The current was resistant to the Na+-channel blockers tetrodotoxin (1 mM) and amiloride (10 mM), while the Ca2+-channel blocker verapamil (50 mM) in the bath solution completely blocked the current. The intracellular Na+ concentration increased in staurosporine-treated oocytes, but could be prevented by replacing extracellular Na+ whith either K+ or Choline(+). Prevention of this influx of Na+ also prevented the STS-induced up-regulation of the caspase-3 activity, suggesting that the intracellular Na+ increase is required to induce apoptosis. Taken together, we have found that a voltage dependent Na+ channel is up-regulated during apoptosis and that influx of Na+ is a crucial step in the apoptotic process in Xenopus oocytes.

Place, publisher, year, edition, pages
Public Library of Science, 2014
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-105899 (URN)10.1371/journal.pone.0088381 (DOI)000332396200017 ()
Available from: 2014-04-14 Created: 2014-04-12 Last updated: 2017-12-05
3. Inhibition of SCN2A ortholog upregulation in Xenopus laevis oocytes prevents cell death
Open this publication in new window or tab >>Inhibition of SCN2A ortholog upregulation in Xenopus laevis oocytes prevents cell death
2014 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Transport of ions across the cell membrane is essential for the regulation of cell death and tissue homeostasis, and alterations in the function of voltage-gated ion channels and of the intracellular ionic compositions interfere with these processes. Opening of K, Na , or Cl channels have been linked to the apoptotic process and in many cases, opening of these channels precede caspase-3 activation and are thus early events in the apoptotic process. Consistent with the role of these channels in apoptosis, inhibition of these channels prevents or delays the apoptotic process. However, the role of ion channels during apoptosis has been difficult to explore, mainly due to unspecific/non-selective ion channe blockers. In the present investigation, the molecular identity of a  voltage-gated Na channel in oocytes from Xenopus laevis, which is crucial for the apoptotic response to mechanical stress, was identified. Specific down regulation of SCN2A Na channel expression by miRNA prevented apoptosis, suggesting that Na+ influx is essential for apoptosis in Xenopus oocytes.

National Category
Cell Biology
Identifiers
urn:nbn:se:liu:diva-111044 (URN)
Available from: 2014-10-06 Created: 2014-10-06 Last updated: 2014-10-06Bibliographically approved

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