Structural Basis for Toxin Resistance of β4-Associated Calcium-activated Potassium (BK) Channels

Abstract

The functional diversity of large conductance Ca 2؉ -and voltage-dependent K ؉ (BK) channels arises mainly from co-assembly of the pore-forming mSlo ␣ subunits with four tissueenriched auxiliary ␤ subunits. The structural basis of the interaction between ␣ subunits with ␤ subunits is not well understood. Using computational and experimental methods, we demonstrated that four mSlo turrets decentralized distally from the channel pore to provide a wide open conformation and that the mSlo and h␤4 subunits together formed a "helmet" containing three basic residues (Lys-120, Arg-121, and Lys-125), which impeded the entry of charybdotoxin (ChTX) by both the electrostatic interaction and limited space. In addition, the tyrosine insert mutant (in100Y) showed 56% inhibition, with a K d ‫؍‬ 17 nM, suggesting that the h␤4 lacks an external ChTX-binding site (Tyr-100). We also found that mSlo had an internal binding site (Tyr-294) in the ␣ subunits that could "permanently" block 15% of mSlo؉h␤4 currents in the presence of 100 nM ChTX. These findings provide a better understanding of the diverse interactions between ␣ and ␤ subunits and will improve the design of channel inhibitors.It is well known that functional diversity of large conductance Ca 2ϩ -and voltage-dependent potassium (BK) channels arises mainly from co-expression of the pore-forming ␣ subunits with the tissue-enriched auxiliary ␤ subunits (1-10). Four mammalian ␤ genes (␤1-␤4) are responsible for a variety of the kinetic and pharmacological characteristics of BK channels in native tissues, even though they share sequence homology. For instance, the brain-enriched h␤4 subunits not only alter the conductance-voltage curve of mSlo channels but also slow both the activation and deactivation time courses, suggesting that ␤4 plays a critical role in the regulation of neuronal excitability and neurotransmitter release (9). Furthermore, results from experiments with a ␤4 knock-out showed reduced dentate gyrus excitability and protection against temporal lobe seizures (11).Natural venomous peptides have been used widely to probe the pore conformation of potassium channels. One particular scorpion toxin, charybdotoxin (ChTX), 4 was used to probe the pore structure of BK channels. It is known that the ␣ϩh␤1 has a K d for ChTX similar to that of mSlo channels (4). However, BK channels associating with the ␤2 or ␤3 subunits usually have about 30-fold lower sensitivity to ChTX in equilibrium (3, 4), whereas the ␣ϩh␤4 channel has about 1000-fold slower association with the toxin (7). Is there a common mechanism working for all of four ␤ genes?To explore the toxin resistance mechanism of h␤4 subunits, Jin et al. (12) investigated how the N-linked glycosylation of the h␤4 subunit modulated the toxin sensitivity of hSloϩh␤4 channels. They confirmed that the double glycosylation site mutated in h␤4 showed reduced protection of the channel against toxin blockade as compared with the hSlo channel co-expressed with the wild type h␤4 subunits. Another study is in regard to the remo...

Ca2+-Activated K+Channels: From Protein Complexes to Function

“…Distinct structure and charge distribution in this region determine the dif ferential effects of the I3K/3s on toxin binding (28, 248). Thus, while coassembly with BK/34 or BK/32/3 strongly reduces ChTX-mediated inhibition of BK< a channels by 1,000-and 30-fold, respectively (59,130,240), channels associated with BK/31 retain their high sensitivity to this blocker (74).…”

Hg1, Novel Peptide Inhibitor Specific for Kv1.3 Channels from First Scorpion Kunitz-type Potassium Channel Toxin Family

“…All of the channels were subcloned into the pIRES2-EGFP vector (Clontech, Mountain View, CA) and transformed into HEK293 cells. The whole-cell patch clamp was used to measure and record the channel currents according to a previously described procedure (13,31). Peptides were dissolved in stock solutions containing 1% BSA and diluted into solutions containing 0.01% BSA for toxin application in electrophysiological experiments.…”

Scorpion Potassium Channel-blocking Defensin Highlights a Functional Link with Neurotoxin

“…The Extracellular Pore Region of the Kv1.3 Channel Serves as the BmKDfsin4-interacting Interface-Given that the potassium channel vestibule acts as the binding site of scorpion toxin blockers (18,19,24) (Fig. 5A), we further investigated whether the Kv1.3 channel extracellular pore region is the BmKDfsin4-interacting interface.…”

“…affinities because of the dominant electrostatic interactions between the basic residues in scorpion toxins and acidic residues in potassium channels (18,19,24). On the basis of the evolutionary function of acidic residues in orienting the toxin binding interface (8,25), the channel affinity effects of five basic residues (Lys-13, Arg-19, Arg-20, Arg-21, and Arg-37), which are physically far from Asp-26 of BmKDfsin4, were investigated (Fig.…”