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Updated: Sep 27, 2026
Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria
Published on: September 7, 2012
Matrix volume measurements challenge the existence of diazoxide/glibencamide-sensitive KATP channels in rat
Manika Das1, Joanne E Parker, Andrew P Halestrap
1Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
Abstract:
A mitochondrial sulphonylurea-sensitive, ATP-sensitive K+ channel (mitoKATP) that is selectively inhibited by 5-hydroxydecanoate (5-HD) and activated by diazoxide has been implicated in ischaemic preconditioning. Here we re-evaluate the evidence for the existence of this mitoKATP by measuring changes in light scattering (A520) in parallel with direct determination of mitochondrial matrix volumes using 3H2O and [14C]sucrose. Incubation of rat liver and heart mitochondria in KCl medium containing Mg2+ and inorganic phosphate caused a decrease in light scattering over 5 min, which was accompanied by a small (15-30 %) increase in matrix volume. The presence of ATP or ADP in the buffer from the start greatly inhibited the decline in A520, whilst addition after a period of incubation (1-5 min) induced a rapid increase in A520, especially in heart mitochondria. Neither response was accompanied by a change in matrix volume, as measured isotopically. However, the effects of ATP and ADP on A520 were abolished by carboxyatractyloside and bongkrekic acid, inhibitors of the adenine nucleotide translocase (ANT) that lock the transporter in two discrete conformations and cause distinct changes in A520 in their own right. These data suggest that rather than matrix volume changes, the effects of ATP and ADP on A520 reflect changes in mitochondrial shape induced by conformational changes in the ANT. Furthermore, we were unable to demonstrate either a decrease in A520 or increase in matrix volume with a range of ATP-sensitive K+ channel openers such as diazoxide. Nor did glibencamide or 5-HD cause any reduction of matrix volume, whereas the K+ ionophore valinomycin (0.2 nM), produced a 10-20 % increase in matrix volume that was readily detectable by both techniques. Our data argue against the existence of a sulphonylurea-inhibitable mitoKATP channel.
Insights
This study challenges the existence of the mitochondrial ATP-sensitive potassium channel (mitoKATP). Researchers found no evidence supporting its role in mitochondrial function or response to common modulators.
Area of Science:
- Mitochondrial Physiology
- Cellular Signaling
- Biophysics
Background:
- The mitochondrial ATP-sensitive potassium channel (mitoKATP) is proposed to be involved in ischemic preconditioning.
- This channel is reportedly inhibited by 5-hydroxydecanoate (5-HD) and activated by diazoxide.
Purpose of the Study:
- To re-evaluate the evidence for the existence of the mitoKATP channel.
- To investigate the effects of ATP, ADP, and specific channel modulators on mitochondrial volume and light scattering.
Main Methods:
- Measurement of light scattering (A520) in isolated rat liver and heart mitochondria.
- Direct determination of mitochondrial matrix volumes using 3H2O and [14C]sucrose.
- Assessment of adenine nucleotide translocase (ANT) activity using specific inhibitors (carboxyatractyloside, bongkrekic acid).
Main Results:
- ATP and ADP induced changes in light scattering, but not matrix volume, suggesting effects on mitochondrial shape via ANT.
- Neither diazoxide nor 5-HD produced expected changes in light scattering or matrix volume.
- Valinomycin caused a detectable increase in matrix volume, serving as a positive control.
Conclusions:
- The data argue against the existence of a sulphonylurea-inhibitable mitoKATP channel.
- Observed effects of ATP/ADP on light scattering are attributed to conformational changes in the ANT, not mitoKATP activity.
- The proposed role of mitoKATP in ischemic preconditioning requires re-evaluation.
