A heme-binding domain controls regulation of ATP-dependent potassium channels

Mark J Burton,Nina M Storey,John S Mitcheson,Tatyana Chernova,Jérôme Santolini,Pierre Dorlet,Noel W Davies,Ralf Schmid,Andrew G Jamieson,Sofia M Kapetanaki,Peter C E Moody,Emma L Raven

doi:10.1073/pnas.1600211113

Abstract

Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATP channels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels.

Highlights

Heme iron has many and varied roles in biology
Heme-containing proteins form a large and biologically important group of enzymes: they are found in all living species and carry out a wide variety of functions, for example, in oxygen transport, electron transfer, and in various hemedependent catalytic processes. For many of these proteins, much structural and mechanistic information is available, and this has led to an established view that the role of heme in biology is as a prosthetic group, which means that it binds tightly to a particular protein, conferring specific properties that vary according to the biological requirements
We find clear evidence for hemedependent modulation of cardiac KATP channels, and our analyses indicate that heme interacts with a cytoplasmic regulatory domain to modulate channel activity

Summary

Results

The KATP family of ion channels responds to intracellular ATP and plays a pivotal role in linking cellular metabolism to excitability [7]. The KATP channels were opened by the channel opener P1075 (10 μM), and the resulting currents were recorded from myocytes in the whole-cell configuration. Application of hemin (500 nM) with ATP (500 μM) revealed a significant increase in channel activity, Popen, to 0.104 ± 0.030, n = 4 (Fig. 1 A, iii and B). The KATP subunits Kir6.2 and SUR2A do not contain any CXXCH cytochrome c-like heme-binding motifs, as identified in the large conductance Ca2+-activated K+ channel (BK channels) [12]. Inside-out patches from HEK293 cells heterologously expressing WT Kir6.2 and SUR2A show KATP channel currents with a robust response to heme. In the presence of ATP (500 μM), the KATP channels had a low open probability (0.023 ± 0.009, n = 9; Fig. 3 A and B) compared with the addition of heme (500 nM) when an increase in open probability is observed (0.109 ± 0.020, n = 9; Fig. 3B). Single channel analysis revealed that both single mutations C628S and H648A had a substantial effect on the KATP channel response to heme

ATP ATP

Discussion

Materials and Methods