Mitochondria Present in Excised Patches From Pancreatic B-cells May Form Microcompartments With ATP-Dependent Potassium Channels

Abstract

Experiments with inside-out patches excised from pancreatic B-cells have yielded evidence that mitochondria are often contained in the cytoplasmic plug protruding into the tip of patch pipette. When intact B-cells were loaded with the fluorescent mitochondrial stain, rhodamine 123, and membrane patches excised from these cells, a green fluorescence could be observed in the lumen at the tip of the patch pipette. The same result was obtained with the mitochondrial stain, MitoTracker Green FM, which is only fluorescent in a membrane-bound state. Furthermore, the open probability of ATP-dependent potassium (K(ATP)) channels in inside-out patches was influenced by mitochondrial fuels and inhibitors. Respiratory substrates like tetramethyl phenylene diamine (2 mM) plus ascorbate (5 mM) or alpha-ketoisocaproic acid (10 mM) reduced the open probability of K(ATP) channels in inside-out patches significantly (down to 57% or 65% of control, respectively). This effect was antagonized by the inhibitor of cytochrome oxidase, sodium azide (5 mM). Likewise, the inhibitor of succinate dehydrogenase, malonate (5 mM), increased the open probability of K(ATP) channels in the presence of succinate (1 mM). However, oligomycin in combination with antimycin and rotenone did not increase open probability. Although it cannot be excluded that these effects result from a direct interaction with the K(ATP) channels, the presence of mitochondria in the close vicinity permits the hypothesis that changes in mitochondrial metabolism are involved, mitochondria and K(ATP) channels thus forming functional microcompartments.

Rotenone selectively occludes sensitivity to hypoxia in rat carotid body glomus cells

“…As described in other systems (Kennedy et al 1999; Montero et al 2000), peripheral mitochondria may form functional units with membrane proteins to delimit submembraneous cytosolic microdomains. These ideas are compatible with previous observations describing the modulation of membrane K + channels by O 2 tension in excised membrane patches (Ganfornina & López‐Barneo, 1991; Riesco‐Fagundo et al 2001; Lewis et al 2002) as these are known to contain mitochondria and other intracellular organelles (Sather et al 1992; Rustenbeck et al 1999). The existence of a separate sensor that confers O 2 sensitivity to the K + channels helps to explain why in some conditions, in which the sensor is possibly detached from the channels, glomus cells lose the responsiveness to hypoxia while maintaining normal electrophysiological properties (López‐Barneo et al 1998).…”

Cellular oxygen sensing by mitochondria: old questions, new insight

“…They concluded that the O 2 -sensing function was not intrinsic to the Kv2.1-Kv9.4 complex, on the basis of the fact that only some transfected cells responded to low O 2 concentration (45). However, an alternative interpretation is based on a recent report by Rustenbeck et al (52), who found that mitochondria were present in excised membrane patches from pancreatic ␤-cells. The possibility that mitochondria may remain associated with ATP-sensitive K ϩ (K ATP ) channels by being dragged into the pipette during inside-out excised patch experiments raises the possibility that such organelles could be involved in the O 2 -sensing response.…”

A plasma membrane-associated glycolytic metabolon is functionally coupled to KATPchannels in pancreatic α and β cells from humans and mice

“…Thus, plasma membrane-compartmentalized PK must be sufficiently close to the K ATP channel to locally deplete MgADP and increase ATP to close the channel. An alternative interpretation of this data is that PK-derived pyruvate is oxidized by residual patch-associated mitochondria that generate ATP and close K ATP channels independently of glycolysis 33 . However, pyruvate and ADP were unable to induce K ATP channel closure, and the presence of ATP synthase inhibitor (1 μg/mL oligomycin) did not affect K ATP channel inhibition by PEP and ADP (Supplemental Figure S1).…”

“…In mammalian cells, glycolysis is known to regulate a variety of plasma membrane ion channels and pumps that exist in regions of high ATP consumption, including K 18 . Cardiac K ATP channels are known to associate with multiple enzymes of lower glycolysis which regulate the channel via the activity of PK 25–28 . However, the demonstration of a complete glycolytic metabolon—involving the communication of substrates and nucleotides between upper and lower glycolysis as well as LDH—remains elusive.…”