Abstract
The large-conductance, calcium- and voltage-activated K+(BK) channel consists of the pore-forming α subunits (BKα) and auxiliary subunits. The auxiliary γ1-3 subunits potently modulate the BK channel by shifting its voltage-dependence of channel activation toward the hyperpolarizing direction by approximately 145 mV (γ1), 100 mV (γ2), and 50 mV (γ3). Mallotoxin is a potent small-molecule BK channel activator. We analyzed the relationship between mallotoxin and the γ subunits in their BK channel-activating effects in membrane patches excised from HEK-293 cells. We found that mallotoxin, when applied extracellularly, shifted the half-activation voltage (V1/2) of BKα channels by −72 mV. The channel-activating effect of mallotoxin was greatly attenuated in the presence of the γ1, γ2, or γ3 subunit, with resultant ΔV1/2 (+/− mallotoxin) values of −9, −28, or −15 mV, respectively. Most examined γ1 mutant subunits antagonized mallotoxin’s channel-activating effect in a manner that was largely dependent on its own modulatory function. However, mallotoxin caused an irreversible functional and structural disengagement of the γ1-F273S mutant from BK channels. We infer that the auxiliary γ subunit effectively interferes with mallotoxin on BK channel modulation via either a direct steric competition or an indirect allosteric influence on mallotoxin’s binding and action on BKα.
Highlights
The mammalian big/large-conductance, voltage- and calcium-activated potassium (BK) channel is widely expressed in various tissues and cell types and plays important roles in many physiological processes, including contractile activity of smooth muscles[1], regulation of neurotransmitter release and neuronal firing[2,3,4,5], and frequency tuning of auditory hair cells[6]
Mallotoxin at 2 μM resulted in a large shift of 72 mallotoxin na V1/2 (mV) in the half-activation voltages (V1/2) of BK channels toward the hyperpolarizing direction, compared to the untreated BKαchannels (V1/2 = 164 ± 3 mV, n = 11) in the virtual absence of [Ca2+]i (Fig. 1a and c; Table 1)
The mallotoxin-induced left shift in the conductance-voltage (G-V) relationship was accompanied by a great deceleration in channel deactivation (e.g., ~10 times decrease in rate at −160 mV), while the rate of channel activation at tested voltages (120–200 mV) was largely unaffected (Fig. 1b,d and e)
Summary
The mammalian big/large-conductance, voltage- and calcium-activated potassium (BK) channel is widely expressed in various tissues and cell types and plays important roles in many physiological processes, including contractile activity of smooth muscles[1], regulation of neurotransmitter release and neuronal firing[2,3,4,5], and frequency tuning of auditory hair cells[6]. The γ1, γ2 and γ3 subunits, all ~35 kDa in size, facilitate BK channel activation by shifting voltage-dependence of channel activation in the hyperpolarizing direction over an exceptionally large range, by approximately 145 mV (γ1), 100 mV (γ2), and 50 mV (γ3 ) in the absence of calcium[8,15]. In addition to their regulation by auxiliary proteins, BK channels are modulated by a variety of endogenous or exogenous small peptide or chemical molecules[16,17,18,19]. Correspondence and requests for materials should be addressed to J.Y. (email: jyan1@ mdanderson.org) www.nature.com/scientificreports/
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