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TASK-5, a potential mitochondrial channel characterised by planar patch-clamp electrophysiology

TASK-5, a potential mitochondrial channel characterised by planar patch-clamp electrophysiology
TASK-5, a potential mitochondrial channel characterised by planar patch-clamp electrophysiology
Since its first identification 17 years ago, TWIK-related acid-sensitive K+ channel 5 (TASK-5) was described as a “non-functional” two-pore domain acid-sensitive potassium (K + ) channel family member as, researchers failed to identify K+ currents on the plasma membrane of transfected cells or oocytes. Evidence that TASK-5 protein shows a putative expression within the inner mitochondrial membrane (IMM) was provided by members of the O’Kelly group in 2012; where in vitro binding assays and immunocytochemistry demonstrated that the channel bound mitochondrial resident proteins and showed co-localisation with mitochondrial markers. The work within this thesis tests the hypothesis that TASK-5 preferentially shows intracellular localisation and may elicit K+ currents in mitochondria. An assumption that was further endorsed by immunocytochemistry followed by co-localisation analyses where transfected cells (with a GFP-tagged TASK-5 DNA construct) were stained with either Mitotracker red or IMM protein complexes.

To achieve electrophysiological characterisation of IMM resident TASK-5, an experimental strategy was designed consisting of isolating mitochondria from cells transfected with a GFPtagged channel DNA construct. Organelles expressing the channel were subsequently sorted by flow cytometry to ensure that mitochondria are selected over cell debris. The IMM was then accessed by an incubation in hypotonic conditions to strip off their outer mitochondrial membrane (OMM) and expose the IMM (mitoplasts). The extent of mitochondrial matrix swell induction and OMM rupture was also investigated by transmission electron microscopy (TEM). Last, planar patch-clamping was performed on mitoplasts over-expressing TASK-5 to investigate whether the channel can elicit K+ currents. This was achieved by administrating a cocktail of channel blockers to mitoplasts and comparing residual current amplitudes between non- and transfected organelles. To determine if the channel of interest was acid-sensitive, transfected mitoplasts were bathed in low pH solution and currents were compared to mitoplasts that did not over-express TASK-5.

This study demonstrates that over-expression of TASK-5 may be linked to K + current differences observed during pharmacological assays performed on mitochondrial membranes, explaining why previous published studies were unable to detect K + currents on plasma membranes. The planar patch-clamp method provides the potential to gain electrophysiological understanding, not only for future mitochondrial ion channel studies, but also for broader range of pharmacological and functional intracellular compartment research.

University of Southampton
Georgiades, Eleni
9f51f383-bae4-400c-b94b-6b2c7b7503a1
Georgiades, Eleni
9f51f383-bae4-400c-b94b-6b2c7b7503a1
De Planque, Maurits
a1d33d13-f516-44fb-8d2c-c51d18bc21ba
O'kelly, Ita
e640f28a-42f0-48a6-9ce2-cb5a85d08c66
Smith, Peter
003de469-9420-4f12-8f0e-8e8d76d28d6c

Georgiades, Eleni (2018) TASK-5, a potential mitochondrial channel characterised by planar patch-clamp electrophysiology. University of Southampton, Doctoral Thesis, 220pp.

Record type: Thesis (Doctoral)

Abstract

Since its first identification 17 years ago, TWIK-related acid-sensitive K+ channel 5 (TASK-5) was described as a “non-functional” two-pore domain acid-sensitive potassium (K + ) channel family member as, researchers failed to identify K+ currents on the plasma membrane of transfected cells or oocytes. Evidence that TASK-5 protein shows a putative expression within the inner mitochondrial membrane (IMM) was provided by members of the O’Kelly group in 2012; where in vitro binding assays and immunocytochemistry demonstrated that the channel bound mitochondrial resident proteins and showed co-localisation with mitochondrial markers. The work within this thesis tests the hypothesis that TASK-5 preferentially shows intracellular localisation and may elicit K+ currents in mitochondria. An assumption that was further endorsed by immunocytochemistry followed by co-localisation analyses where transfected cells (with a GFP-tagged TASK-5 DNA construct) were stained with either Mitotracker red or IMM protein complexes.

To achieve electrophysiological characterisation of IMM resident TASK-5, an experimental strategy was designed consisting of isolating mitochondria from cells transfected with a GFPtagged channel DNA construct. Organelles expressing the channel were subsequently sorted by flow cytometry to ensure that mitochondria are selected over cell debris. The IMM was then accessed by an incubation in hypotonic conditions to strip off their outer mitochondrial membrane (OMM) and expose the IMM (mitoplasts). The extent of mitochondrial matrix swell induction and OMM rupture was also investigated by transmission electron microscopy (TEM). Last, planar patch-clamping was performed on mitoplasts over-expressing TASK-5 to investigate whether the channel can elicit K+ currents. This was achieved by administrating a cocktail of channel blockers to mitoplasts and comparing residual current amplitudes between non- and transfected organelles. To determine if the channel of interest was acid-sensitive, transfected mitoplasts were bathed in low pH solution and currents were compared to mitoplasts that did not over-express TASK-5.

This study demonstrates that over-expression of TASK-5 may be linked to K + current differences observed during pharmacological assays performed on mitochondrial membranes, explaining why previous published studies were unable to detect K + currents on plasma membranes. The planar patch-clamp method provides the potential to gain electrophysiological understanding, not only for future mitochondrial ion channel studies, but also for broader range of pharmacological and functional intracellular compartment research.

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Eleni Georgiades thesis FINAL - Version of Record
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Published date: July 2018

Identifiers

Local EPrints ID: 433951
URI: http://eprints.soton.ac.uk/id/eprint/433951
PURE UUID: 21a3d220-c2eb-4f0b-ab2e-a314ffd88901
ORCID for Maurits De Planque: ORCID iD orcid.org/0000-0002-8787-0513
ORCID for Peter Smith: ORCID iD orcid.org/0000-0003-4400-6853

Catalogue record

Date deposited: 09 Sep 2019 16:30
Last modified: 10 Sep 2019 00:41

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Contributors

Author: Eleni Georgiades
Thesis advisor: Maurits De Planque ORCID iD
Thesis advisor: Ita O'kelly
Thesis advisor: Peter Smith ORCID iD

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