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Electrostatic domino effect in the Shaker K channel turret
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
2007 (English)In: Biophysical Journal, ISSN 0006-3495, Vol. 93, no 7, 2307-2314 p.Article in journal (Refereed) Published
Abstract [en]

Voltage-gated K channels are regulated by extracellular divalent cations such as Mg2+ and Sr2+, either by screening of fixed negative surface charges, by binding directly or close to the voltage sensor, or by binding to the pore. Different K channels display different sensitivity to divalent cations. For instance, 20 mM MgCl2 shifts the conductance versus voltage curve, G(V), of the Kv1-type Shaker channel with 14 mV, while the G(V) of Kv2.1 is shifted only with 7 mV. This shift difference is paralleled with different working ranges. Kv1-type channels open at −20 mV and Kv2.1 channel open at +5 mV. The aim of this study was to identify critical residues for this Mg2+-induced G(V) shift by introducing Kv2.1 channel residues in the Shaker K channel. The K channels were expressed in Xenopus laevis oocytes and studied with the two-electrode voltage-clamp technique. We found that three neutral-to-positive amino-acid residue exchanges in the extracellular loops connecting transmembrane segments S5 and S6 transferred the Mg2+-shifting properties. The contributions of the three residues were additive, and thus independent of each other, with the contributions in the order 425 > 419 > 451. Charging 425 and 419 not only affect the Mg2+-induced G(V) shift with 5–6 mV, but also shifts the G(V) with 17 mV. Thus, a few strategically placed surface charges clearly modulate the channel’s working range. Residue 425, located at some distance away from the voltage sensor, was shown to electrostatically affect residue K427, which in turn affects the voltage sensor S4—thus, an electrostatic domino effect.

Place, publisher, year, edition, pages
2007. Vol. 93, no 7, 2307-2314 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-12996DOI: 10.1529/biophysj.107.104349OAI: oai:DiVA.org:liu-12996DiVA: diva2:17635
Available from: 2008-03-10 Created: 2008-03-10 Last updated: 2009-04-24
In thesis
1. Molecular aspects on voltage-sensor movement
Open this publication in new window or tab >>Molecular aspects on voltage-sensor movement
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Voltage-gated ion channels are fundamental for electrical signaling in living cells. They are composed of four subunits, each holding six transmembrane helices, S1-S6. Each subunit contains a voltage-sensor domain, S1-S4, and a pore domain, S5-S6. S4 contains several positively charged amino-acid residues and moves in response to changes in membrane voltage. This movement controls the opening and closing of the channel. The structure of the pore domain is solved and demonstrates principles of channel selectivity. The molecular mechanism of how the voltage sensor regulates the opening of the channel is still under discussion. Several models have been discussed. One of the models is the paddle model where S3b and S4 move together. The second one is the helical-twist where S4 makes a small rotation in order for the channel to open. The third one is the helical-screw model where S4 twists around its axis and moves diagonally towards the extracellular side of the channel.

The aim of this PhD project was to study the molecular movement of the voltage sensor in the depolarization-activated Shaker K channel. Cloned channels were expressed in Xenopus laevis oocytes, and investigated with several electrophysiological techniques.

1. We show that S4 moves in relation to both S3b and S5. The formation of some disulfide bonds between S4 and neighboring positions, in only the open state, shows that the paddle model cannot be correct. Furthermore, electrostatic and steric effects of residues in S3b suggest that S3b is tilted, with the intracellular part close to S4.

2. We show that the relatively Mg-sensitive Shaker K channel is changed into the less Mg-sensitive Kv2.1 K channel with respect to its sensitivity to extracellularly applied Mg2+ by changing the charge of three extracellularly positioned amino acid residues. One of the residues, F425C, mediates its effect through the neighboring residue K427.

3. We show that oxaliplatin, an anti-cancer drug, has no effect on the Shaker K channel. It has been suggested that a negatively charged monochloro complex of oxaliplatin is the active substance, and also causes the neurotoxic side effects. Neither this complex shows any effect on the channel.

Our experiments point towards the helical-screw model. The other models for voltage-sensor movements are incompatible with the results in this study.

Place, publisher, year, edition, pages
Institutionen för klinisk och experimentell medicin, 2007
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1028
Keyword
Potassium channels, voltage-gated, chemistry, metabolism, physiology, patch-clamp techniques, oocytes
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-11229 (URN)978-91-85895-53-3 (ISBN)
Public defence
2007-11-30, Linden, Campus US, Linköpings universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2008-03-10 Created: 2008-03-10 Last updated: 2009-06-10

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Broomand, AmirElinder, Fredrik

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