Connexin 43 impacts on mitochondrial potassium uptake

Kerstin Boengler,Rainer Schulz,Luc Leybaert,Gerd Heusch,Elvira Ungefug

doi:10.3389/fphar.2013.00073

Kerstin Boengler, Rainer Schulz + Show 3 more

Open Access

https://doi.org/10.3389/fphar.2013.00073

Copy DOI

Abstract

In cardiomyocytes, connexin 43 (Cx43) forms gap junctions and unopposed hemichannels at the plasma membrane, but the protein is also present at the inner membrane of subsarcolemmal mitochondria (SSM). Both inhibition and genetic ablation of Cx43 reduce ADP-stimulated complex 1 respiration. Since mitochondrial potassium influx impacts on oxygen consumption, we investigated whether or not inhibition or ablation of mitochondrial Cx43 alters mitochondrial potassium uptake. SSM were isolated from rat left ventricular myocardium and loaded with the potassium-sensitive dye PBFI (potassium-binding benzofuran isophthalate). Intramitochondrial potassium was replaced by tetraethylammonium. Mitochondria were incubated under control conditions or treated with 250 μM Gap19, a peptide that specifically inhibits Cx43-based hemichannels at plasma membranes. Subsequently, 140 mM KCl was added and the slope of the increase in PBFI fluorescence over time was calculated. The slope of the PBFI fluorescence of the control mitochondria was set to 100%. In the presence of Gap19, the mitochondrial potassium influx was reduced from 100 ± 11.6% in control mitochondria to 65.5 ± 10.7% (n = 6, p < 0.05). In addition to the pharmacological inhibition of Cx43, potassium influx was studied in mitochondria isolated from conditional Cx43 knockout mice. Here, the ablation of Cx43 was achieved by the injection of 4-hydroxytamoxifen (4-OHT; Cx43Cre-ER(T)/fl + 4-OHT). The mitochondria of the Cx43Cre-ER(T)/fl + 4-OHT mice contained 3 ± 1% Cx43 (n = 6) of that in control mitochondria (100 ± 11%, n = 8, p < 0.05). The ablation of Cx43 (n = 5) reduced the velocity of the potassium influx from 100 ± 11.2% in control mitochondria (n = 9) to 66.6 ± 5.5% (p < 0.05). Taken together, our data indicate that both pharmacological inhibition and genetic ablation of Cx43 reduce mitochondrial potassium influx.

Highlights

Connexin 43 (Cx43) forms gap junctions between adjacent cardiomyocytes and is essential for cell–cell communication
The mice were sacrificed on day 11 after the first injection and mitochondria were isolated from the left ventricles. 4-OHT-treated Cx43fl/fl mice served as a control for potassium measurements, and untreated Cx43fl/fl mice were used as a control for Western blot analysis
The velocity of the mitochondrial potassium influx was measured in wild-type subsarcolemmal mitochondria (SSM) under control conditions and after incubation with 250 μM Gap19, a peptide that inhibits connexin 43 (Cx43)-based hemichannels at plasma membranes, at excitation wavelengths of 340 and 380 nm, respectively (Figure 1)

Summary

Introduction

Connexin 43 (Cx43) forms gap junctions between adjacent cardiomyocytes and is essential for cell–cell communication. Six Cx43 proteins at the plasma membrane assemble into a hemichannel, and hemichannels open during ischemia and thereby contribute to cell injury (Shintani-Ishida et al, 2007; Clarke et al, 2009). Cx43 is present at the inner membrane of cardiomyocyte mitochondria, and cross-linking studies suggest the presence of Cx43-hemichannels within cardiomyocyte mitochondria (Miro-Casas et al, 2009). In contrast to subsarcolemmal mitochondria (SSM), interfibrillar mitochondria (IFM) lack Cx43 (Boengler et al, 2009). An analysis of the impact of Cx43 on mitochondrial function revealed reduced oxygen consumption in mitochondria in which Cx43 was either inhibited by 18αglycyrrhetinic acid (18αGA) or Cx43-mimetic peptides or in which Cx43 was deleted by conditional knockout (Boengler et al, 2012)

Objectives

Methods

Results

Conclusion