The External TEA Binding Site and C-Type Inactivation in Voltage-Gated Potassium Channels

Abstract

The location of the tetraethylammonium (TEA) binding site in the outer vestibule of K+ channels, and the mechanism by which external TEA slows C-type inactivation, have been considered well-understood. The prevailing model has been that TEA is coordinated by four amino acid side chains at the position equivalent to Shaker T449, and that TEA prevents a constriction that underlies inactivation via a foot-in-the-door mechanism at this same position. However, a growing body of evidence has suggested that this picture may not be entirely correct. In this study, we reexamined these two issues, using both the Kv2.1 and Shaker potassium channels. In contrast to results previously obtained with Shaker, substitution of the tyrosine at Kv2.1 position 380 (equivalent to Shaker 449) with a threonine or cysteine had a relatively minor effect on TEA potency. In both Kv2.1 and Shaker, modification of cysteines at position 380/449 by 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET) proceeded at identical rates in the absence and presence of TEA. Additional experiments in Shaker demonstrated that TEA bound well to C-type inactivated channels, but did not interfere with MTSET modification of C449 in inactivated channels. Together, these findings rule out the possibility that TEA binding involves an intimate interaction with the four side chains at the position equivalent to Shaker 449. Moreover, these results argue against the model whereby TEA slows inactivation via a foot-in-the-door mechanism at position 449, and also argue against the hypothesis that the position 449 side chains move toward the center of the conduction pathway during inactivation. Occupancy by TEA completely prevented MTSET modification of a cysteine in the outer-vestibule turret (Kv2.1 position 356/Shaker position 425), which has been shown to interfere with both TEA binding and the interaction of K+ with an external binding site. Together, these data suggest that TEA is stabilized in a more external position in the outer vestibule, and does not bind via direct coordination with any specific outer-vestibule residues.

Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1

“…Among tested ligands, only 4AP did not compete with R-AgTx2 for binding to the pore region of KcsA-Kv1.1, because 4AP binds to the site located on the inner side of the channel pore (44,45). Another small organic molecule TEA can bind to potassium channels on either side (43). These results provide confirmatory evidence that the hybrid KcsA-Kv1.1 channel possesses, in general, a Kv1.1 ligand affinity profile, and that the novel test system based on KcsA-Kv1.1-expressing spheroplasts can be used for toxin screening.…”

Voltage-Gated K+ Channel Block by Catechol Derivatives: Defining Nonselective and Selective Pharmacophores

“…Indeed, a similar interaction with a bacterial channel has been modeled with molecular dynamics (Crouzy et al, 2001;Guidoni and Carloni, 2002;Luzhkov et al, 2003) and studied by crystallization with TEA analogs (Lenaeus et al, 2005). However, site-directed mutagenesis and chemical modification results have called this model into question for Kv2.1: although the tyrosine side chain clearly has a role in external TEA block, it may not cage or bind TEA stably or directly in mammalian Kv2.1 channels (Pascual et al, 1995;Andalib et al, 2004). The uncertainty in the mechanism of the TEA block of Kv2.1 makes interpretation of the competition between TEA and 48F10, but not catechol and many derivatives, difficult to interpret in structural terms.…”

1,4-Diazabicyclo[2.2.2]octane Derivatives: A Novel Class of Voltage-Gated Potassium Channel Blockers

“…According to some reports, Kv2.1 residue Tyr380 (equivalent to KcsA Tyr82 and Shaker Thr449) in the outer pore (Fig. 9C) forms at least part of the external TEA binding site (MacKinnon and Yellen, 1990;Heginbotham and MacKinnon, 1992;Luzhkov and Aqvist, 2001) although this has been subsequently disputed (Pascual et al, 1995;Crouzy et al, 2001;Andalib et al, 2004). The finding that Kv channels with an aromatic residue in this position are highly sensitive to block by external TEA was suggested to explain the higher TEA affinity of wild-type Kv2.1 compared with wild-type Shaker (MacKinnon and Yellen, 1990).…”

“…Methanethiosulfonate forms the basis for a group of compounds that are able to bind irreversibly to the free sulfhydryl group of cysteine residues that line the pore of channels (Akabas et al, 1992;Karlin and Akabas, 1998). As was demonstrated by the Korn group (Andalib et al, 2004), introduction of a cysteine at position 356 in Kv2.1 made this position modifiable with MTSET, resulting in permanent fractional block. However, when TEA was administered before MTSET, this ability to permanently modify the introduced cysteine was lost.…”

“…This was first proposed to result from cation-orbital interactions but later suggested to arise from differences in the hydration state of TEA (Crouzy et al, 2001). In a more recent study, however, an external binding site more distal to the selectivity filter was proposed, based on the ability of TEA to protect a cysteine introduced at position 356 in Kv2.1 from modification by methanethiosulfonate ethyltrimethylammonium (MT-SET), which normally manifests as permanent channel block (Andalib et al, 2004).…”