Enzymatic Activity of the Human BK Channel: A Function Beyond Electrical Signaling

Abstract

BK (Slo1) channels open probability is acutely regulated by heme, which associates with their intracellular multi-ligand sensor, the gating ring. We found that the gating ring region encompassing the heme-binding site shares structural homology with Cytochrome C (CytC), the well-known hemoprotein. In addition to its role in electron shuttling, CytC exhibits various catalytic properties such as peroxidase activity, i.e. the oxidation of suitable substrates using peroxides. To probe for peroxidase activity of the CytC-like domain in a purified BK channel gating ring, we used the chromophore 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the oxidizable substrate. We found that the BK gating ring complexed with heme in the presence of H2O2 catalyzes the formation of ABTS+• cation radical, monitored by absorption at 415nm. The initial rate of ABTS+• formation was linearly correlated with [gating ring] between 0.05-0.3 μM. Disruption of the heme regulatory motif (C615S/H616R) significantly decreased the initial rate of ABTS+• formation. The kinetic parameters of the enzymatic reaction were determined by performing two-substrate Michaelis-Menten analysis, which yielded for gating ring: kcat/KmH2O2 ≈12 s−1mM−1 and kcat/KmABTS ≈0.5 s−1mM−1. For CytC, we estimated kcat/KmH2O2 ≈1.4 s−1mM−1 and kcat/KmABTS ≈0.035 s−1mM−1. These results suggest that, under our experimental conditions, the gating ring catalytic efficiency is ∼10 times higher than CytC. Finally, we found that HEK cells expressing BK channels (blocked with 100 nM Iberiotoxin) are significantly more resistant to oxidative insult (200 μM H2O2) than cells expressing BK channels with impared heme binding (C615S/H616R) (p<0.05) as revealed by the increased cell viability (MTT assay). Thus, the BK channel exhibits peroxidase activity and confers a protective effect against oxidative cell damage. These results redefine the role of BK channels, assigning a catalytic property, in addition to their established K+ conducting properties.