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Quantification of the gating charge in K+ and Na+ ion channels by use of eGFP fluorescence


Grögler, D. Quantification of the gating charge in K+ and Na+ ion channels by use of eGFP fluorescence. 2009, University of Zurich, Faculty of Science.

Abstract

Ion channels are vital for nearly every form of life as we know it. Embedded in the cellular membrane, these complex macromolecular proteins control ionic conductivity by opening or closing an ionic pore upon mechanical, chemical or electrical stimuli. In nerve cells, voltage gated ion channels generate and mediate the transduction of electrical signals from one cell to the next. Some residues of the channel structure are electrically charged and can move through the membrane in response to changes in the transmembrane voltage. These charges are called gating charges. As a consequence of their movement, the three–dimensional conformation of the protein changes and triggers the opening or closing of the ionic pore. The interplay of sodium– and potassium–selective ion channels (Na+ and K+ channels) in nerve cells gives rise to the so–called action potential. The voltage dependence of the probability of channels to open their ionic pore is so steep that a change of the transmembrane voltage of as little as 10mV can increase this probability by a factor of 150. For Shaker type K+ channels, the gating charge has consistently been reported to be of the size of 13 elementary charges. However, for Na+ channels corresponding findings vary between 4 and 16 elementary charges. The gating current is the current due to the movement of the gating charges in the electric field along the ionic pore. For a single voltage gated ion channel the gating current is in the order of pA and therefore below detection threshold in complex biological systems. In the present study, the gating charges of K+ and Na+ ion channels have been determined by the controlled electrical stimulation of cells containing 108–1010 channel proteins. The number of channels was quantified by a novel approach based on fluorescence intensitymeasurements of genetically modified ion channels labeled with enhanced green fluorescent proteins (eGFP). On the basis of the known value for the gating charge of the K+ channel, it has been possible to determine that the size of the gating charge in the rBIIA Na+ channel is of 5.2±0.3 elementary charges. The present thesis is structured in three parts: 1) An introductory chapter on voltage gated ion channels, 2) an experimental part dealing with the determination of the gating charge of the Shaker K+ channel and of the rBIIA Na+ channel, including a comparison of both gating charges and 3) a chapter on the use of optical fibers to locally excite fluorescent proteins and detect their fluorescence emission light. The introductory part is intended for the reader to get a brief overview about the biological significance of voltage gated ion channels for living organisms, about their function and their molecular structure. The relation between the structure and the function of voltage gated ion channels is discussed in the context of the most important publications of the last 20 years. In order to provide the necessary background for the second chapter, the primary focus was laid on the gating process of voltage gated ion channels and its structural implications. The second chapter deals with experiments on the quantification of the gating charge in Shaker K+ channels and rBIIA Na+ channels. The gating charge of a large number of K+ and Na+ channels was determined and the number of channels was estimated by fluorescence intensity measurements. The experiments presented in this chapter have led to the publication More gating charges are needed to open a Shaker K+ channel than are needed to open a rBIIA Na+ channel, Biophysical Journal, 95(3):1165–1175, 2008. The strong autofluorescence of the specimen handicapped the fluorescence intensity measurements in the preceding experiments. To limit the excitation volume – and thereby reduce the autofluorescence signal – a system was developed that uses optical fibers both to excite fluorophores and to detect their fluorescence emission. In the third chapter this fiber optical system is introduced and preliminary experiments on the fluorescence properties of enhanced green fluorescent proteins are presented.

Abstract

Ion channels are vital for nearly every form of life as we know it. Embedded in the cellular membrane, these complex macromolecular proteins control ionic conductivity by opening or closing an ionic pore upon mechanical, chemical or electrical stimuli. In nerve cells, voltage gated ion channels generate and mediate the transduction of electrical signals from one cell to the next. Some residues of the channel structure are electrically charged and can move through the membrane in response to changes in the transmembrane voltage. These charges are called gating charges. As a consequence of their movement, the three–dimensional conformation of the protein changes and triggers the opening or closing of the ionic pore. The interplay of sodium– and potassium–selective ion channels (Na+ and K+ channels) in nerve cells gives rise to the so–called action potential. The voltage dependence of the probability of channels to open their ionic pore is so steep that a change of the transmembrane voltage of as little as 10mV can increase this probability by a factor of 150. For Shaker type K+ channels, the gating charge has consistently been reported to be of the size of 13 elementary charges. However, for Na+ channels corresponding findings vary between 4 and 16 elementary charges. The gating current is the current due to the movement of the gating charges in the electric field along the ionic pore. For a single voltage gated ion channel the gating current is in the order of pA and therefore below detection threshold in complex biological systems. In the present study, the gating charges of K+ and Na+ ion channels have been determined by the controlled electrical stimulation of cells containing 108–1010 channel proteins. The number of channels was quantified by a novel approach based on fluorescence intensitymeasurements of genetically modified ion channels labeled with enhanced green fluorescent proteins (eGFP). On the basis of the known value for the gating charge of the K+ channel, it has been possible to determine that the size of the gating charge in the rBIIA Na+ channel is of 5.2±0.3 elementary charges. The present thesis is structured in three parts: 1) An introductory chapter on voltage gated ion channels, 2) an experimental part dealing with the determination of the gating charge of the Shaker K+ channel and of the rBIIA Na+ channel, including a comparison of both gating charges and 3) a chapter on the use of optical fibers to locally excite fluorescent proteins and detect their fluorescence emission light. The introductory part is intended for the reader to get a brief overview about the biological significance of voltage gated ion channels for living organisms, about their function and their molecular structure. The relation between the structure and the function of voltage gated ion channels is discussed in the context of the most important publications of the last 20 years. In order to provide the necessary background for the second chapter, the primary focus was laid on the gating process of voltage gated ion channels and its structural implications. The second chapter deals with experiments on the quantification of the gating charge in Shaker K+ channels and rBIIA Na+ channels. The gating charge of a large number of K+ and Na+ channels was determined and the number of channels was estimated by fluorescence intensity measurements. The experiments presented in this chapter have led to the publication More gating charges are needed to open a Shaker K+ channel than are needed to open a rBIIA Na+ channel, Biophysical Journal, 95(3):1165–1175, 2008. The strong autofluorescence of the specimen handicapped the fluorescence intensity measurements in the preceding experiments. To limit the excitation volume – and thereby reduce the autofluorescence signal – a system was developed that uses optical fibers both to excite fluorophores and to detect their fluorescence emission. In the third chapter this fiber optical system is introduced and preliminary experiments on the fluorescence properties of enhanced green fluorescent proteins are presented.

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Additional indexing

Item Type:Dissertation
Referees:Fink H W, Greeff K, Robmann P
Communities & Collections:07 Faculty of Science > Physics Institute
Dewey Decimal Classification:530 Physics
Language:English
Date:2009
Deposited On:12 Mar 2010 09:08
Last Modified:05 Apr 2016 14:03
Number of Pages:117
Related URLs:http://opac.nebis.ch/F/?local_base=NEBIS&con_lng=GER&func=find-b&find_code=SYS&request=005859814

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