Molecular modeling of a tandem two pore domain potassium channel reveals a putative binding site for general anesthetics

Abstract

Anesthetics are thought to mediate a portion of their activity via binding to and modulation of potassium channels. In particular, tandem pore potassium channels (K2P) are transmembrane ion channels whose current is modulated by the presence of general anesthetics and whose genetic absence has been shown to confer a level of anesthetic resistance. While the exact molecular structure of all K2P forms remains unknown, significant progress has been made toward understanding their structure and interactions with anesthetics via the methods of molecular modeling, coupled with the recently released higher resolution structures of homologous potassium channels to act as templates. Such models reveal the convergence of amino acid regions that are known to modulate anesthetic activity onto a common three- dimensional cavity that forms a putative anesthetic binding site. The model successfully predicts additional important residues that are also involved in the putative binding site as validated by the results of suggested experimental mutations. Such a model can now be used to further predict other amino acid residues that may be intimately involved in the target-based structure-activity relationships that are necessary for anesthetic binding.

Keywords: Tandem pore potassium channel; anesthesia; homology modeling.

Figure 1

Multiple sequence alignment of the two templates with LyTASK. L159 and the ILRFLT sequences are outlined in boxes. Note the differences in the aligned amino acids between LyTASK and 3UKM/TWIK (both anesthetic sensitive) and 3UM7/TRAAK which is anesthetic insensitive. Amino acid similarity is denoted by shades of blue, with darker indicating greater similarity. Note also the alpha helical structure identified by the Kabsh and Sander algorithm for each structure indicated by the orange bars.

Figure 2

Molecular modeling derived from the consensus overlap of templates reveals a putative general anesthetic binding site. Model of LyTASK illustrating the positions of two established determinants of anesthetic sensitivity: L159 and the critical sequence of ILRFLT amino acids.^,, The putative anesthetic binding pocket is shown by the pink surface, but its intracellular extent is rather arbitrary due to its “cave-like” opening in that direction.

Figure 3

Model of LyTASK derived from the consensus overlap of templates illustrating the position of L159 relative to other adjacent residues for possible mutational analyses. In particular, note the positions of L241 and L242. Distances between respective α carbon atoms are expressed in angstroms. Also notice the distances from R245 to both S155 and Q156 for possible polar interactions.

Figure 4

Expanded view (cylinder rotated 90°) of a possible anesthetic binding region, as viewed from the intracellular surface (blue arrow), illustrating the side chain intercalation of LEU 241 and 242 from one α helix with LEU 159 of the adjacent α helix.

Figure 5

Typical electrophysiology current–voltage relations for the WT and mutant LyTASK potassium channels. (A) Schematic diagram showing current–voltage relation for an untransfected cell (green line), LyTASK transfected cells exhibit a large outwardly rectifying potassium current (blue line) reversing close to −90 mV (R). In the presence of halothane (red line), the LyTASK current is increased. LyTASK currents are quantified by measuring the value of the current at a membrane potential of −50 mV, marked on the diagram is the control LyTASK current “L” and the halothane-activated LyTASK current, “H”. (B) Wild-type LyTASK. (C) LyTASK S155A mutant. (D) LyTASK L242A mutant. (E) LyTASK S155W mutant. (F) LyTASK L241A mutant. Solid lines are LyTASK currents in the absence of halothane, and dashed lines are the LyTASK currents in the presence of 3% halothane. Data were sampled at 20 kHz, and each trace contains 3000 data points. Lines shown are means of 10 individual voltage ramps for a given cell in each condition.

Figure 6

Effect of point mutations in LyTASK currents on (A) baseline control current at −50 mV (“L” in Figure 5A), (B) halothane activated current at −50 mV (“H” in Figure 5A), and (C) percentage activation by halothane. Percentage activation is calculated using (H/L – 1) × 100%. Values shown are means, and error bars are SEM (n = 7 WT, n = 4 L241A, n = 7 L242A, n = 5 S155A, n = 5 S155W). ***p < 0.001, **p < 0.01, *p < 0.05 compared to WT; one-way ANOVA with Bonferroni’s post hoc test.