Heme-Driven Conformational Changes in the Human Slo1 BKCa Channel Gating Ring

Large-conductance voltage- and Ca2+-activated K+ channels (BKCa) are regulated by signaling molecules acting on two intracellular Regulator of K+ Conductance (RCK) domains which assemble into a gating ring superstructure. The discovery of heme as a BKCa channel modulator has unveiled novel aspects of its physiological function (Tang et al., Nature 2003). A recent study (Horrigan et al., JGP 2005) suggests that heme induces conformational changes within the BKCa gating ring altering its interaction with the voltage sensor.Here, we report direct evidence of heme-induced conformational transitions in the purified human BKCa gating ring, expressed and purified from E. coli as reported previously. Its assembly into physiologically-relevant homotetramers (∼310kDa) was confirmed by size-exclusion chromatography. Heme-binding properties of the gating ring were studied under physiologically-relevant conditions using steady-state and time-resolved fluorescence spectroscopy in combination with biochemical methods. Heme binding to BKCa gating ring was detected using UV-visible absorption spectroscopy, which revealed a strong peak at 420nm (Soret band) and a peak at 550nm (α/β-band), characteristic of heme-protein complex formation. The gating ring's Tryptophan fluorescence decreased by up to 44±1.5% with the addition of heme in a dose-dependent manner (K0.5=211±24.0nM, n=2.7±0.10). In contrast, addition of protoporphyrin IX (900 nM), a heme analog lacking Fe, did not affect the Tryptophan fluorescence intensity. Time-correlated single-photon counting (TCSPC) spectroscopy resolved heme-induced structural alterations of the gating ring. Heme binding reduced the average excited-state Tryptophan lifetime from ∉"avg=2.6ns (no heme) to ∉"avg=1.6ns ([heme]=687nM), revealing an altered conformational state. Effects of heme and Ca2+ on fluorescence lifetime were not additive as, after saturating Ca2+-induced effect were reached (35∈1/4M), addition of saturating [heme]=687nM produced minimal effect (∉"avg=1.5ns). In summary, we demonstrate heme-induced conformational transitions in the human BKCa gating ring, likely correlated to the modulation of channel activity.