Abstract
Voltage-gated potassium (KV) channels can be opened by negatively charged resin acids and their derivatives. These resin acids have been proposed to attract the positively charged voltage-sensor helix (S4) toward the extracellular side of the membrane by binding to a pocket located between the lipid-facing extracellular ends of the transmembrane segments S3 and S4. By contrast to this proposed mechanism, neutralization of the top gating charge of the Shaker KV channel increased resin-acid-induced opening, suggesting other mechanisms and sites of action. Here, we explore the binding of two resin-acid derivatives, Wu50 and Wu161, to the activated/open state of the Shaker KV channel by a combination of in silico docking, molecular dynamics simulations, and electrophysiology of mutated channels. We identified three potential resin-acid-binding sites around S4: (1) the S3/S4 site previously suggested, in which positively charged residues introduced at the top of S4 are critical to keep the compound bound, (2) a site in the cleft between S4 and the pore domain (S4/pore site), in which a tryptophan at the top of S6 and the top gating charge of S4 keeps the compound bound, and (3) a site located on the extracellular side of the voltage-sensor domain, in a cleft formed by S1-S4 (the top-VSD site). The multiple binding sites around S4 and the anticipated helical-screw motion of the helix during activation make the effect of resin-acid derivatives on channel function intricate. The propensity of a specific resin acid to activate and open a voltage-gated channel likely depends on its exact binding dynamics and the types of interactions it can form with the protein in a state-specific manner.
Highlights
Resin acids, which are primarily found in pine resin, and their chemical derivatives promote the opening of several voltage-gated potassium (KV) channels (Ottosson et al, 2015, 2017, 2014; Sakamoto et al, 2017; Salari et al, 2018; Silverå Ejneby et al, 2018) and the voltagegated and calcium-activated BK channel (Imaizumi et al, 2002; Sakamoto et al, 2006)
The 2R motif increased the maximum G(V) shift of three resin-acid derivatives In previous work we suggested that resin-acid derivatives open the Shaker KV channel by binding to the S3/S4 cleft (Fig. 1A,B; Ottosson et al, 2017)
The mechanism we proposed for this increased shift presumably involves a direct interaction of M356R and A359R with the compound in the activated/open state, and a stabilization of this state relative to the resting/intermediate closed ones (Ottosson et al, 2014, Fig. 1 A,B)
Summary
Resin acids, which are primarily found in pine resin, and their chemical derivatives promote the opening of several voltage-gated potassium (KV) channels (Ottosson et al, 2015, 2017, 2014; Sakamoto et al, 2017; Salari et al, 2018; Silverå Ejneby et al, 2018) and the voltagegated and calcium-activated BK channel (Imaizumi et al, 2002; Sakamoto et al, 2006). The negatively charged resin acid is suggested to stabilize the activated state of the voltage-sensor domain (VSD) in which the positively charged S4 helix is in an up state, thereby promoting gate opening in the pore domain of the channel. Another equivalent way to describe this effect is to consider that the resin-acid derivative exerts an upward and clockwise electrostatic pull on S4 (Fig 1C; Ottosson et al, 2014, 2015, 2017; Silverå Ejneby et al, 2018). The G(V) shift and the GMAX increase can be caused by a common site and mechanism of action, but, at least for polyunsaturated fatty acids, which share some functional properties with the resin acids (Ottosson et al, 2014), it has been suggested that these two effects on the cardiac KV7.1 channel are mediated via two different parts of the channel (Liin et al, 2018b)
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