Permeation mechanisms through the selectivity filter and the open helix bundle crossing gate of GIRK2

Abstract

G protein-gated inwardly rectifying potassium channels (GIRK) are essential for the regulation of cellular excitability, a physiological function that relies critically on the conduction of K⁺ ions, which is dependent on two molecular mechanisms, namely selectivity and gating. Molecular Dynamics (MD) studies have shown that K⁺ conduction remains inefficient even with open channel gates, therefore further detailed study on the permeation events is required. In this study, all-atom MD simulations were employed to investigate the permeation mechanism through the GIRK2 selectivity filter (SF) and its open helix bundle crossing (HBC) gate. Our results show that it is the SF rather than the HBC or the G-loop gate that determines the permeation efficiency upon activation of the channel. SF-permeation is accomplished by a water-K⁺ coupled mechanism and the entry to the S1 coordination site is likely affected by a SF tilt. Moreover, we show that a 4-K⁺ occupancy in the SF-HBC cavity is required for the permeation through an open HBC, where three K⁺ ions around E152 help to abolish the unfavorable cation-dipole interactions that function as an energy barrier, while the fourth K⁺ located near the HBC allows for the inward transport. These findings facilitate further understanding of the dynamic permeation mechanisms through GIRK2 and potentially provide an alternative regulatory approach for the Kir3 family given the overall high evolutionary residue conservation.

Keywords: G protein-gated inwardly rectifying K+ 2 (GIRK2), permeation mechanism; Helix bundle crossing gate; Inward rectifier potassium (Kir) channel; Molecular dynamics.

Graphical abstract

Fig. 1

Two possible permeation mechanisms through the selectivity filter of ion channels. The related channels are indicated at the bottom of each mechanism. The GIRK2 channel that is involved in both permeation mechanisms requires further clarification.

Fig. 2

K⁺ ions permeating the GIRK2 channel from the extracellular to the intracellular side. K⁺ ions are unable to pass through the channel in a closed conformation. Unexpectedly, K⁺ ions are unable to permeate an open HBC/G-loop gates of GIRK2 even with the aid of an external electric field, suggesting there may be an additional requirement for the permeation in addition to the channel conductive state.

Fig. 3

Computational protocols for simulations, including two major stages colored in yellow and cyan individually. The details of each step are outlined in the Materials and Methods section. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

The number of water molecules for hydration right before and after the permeant movement of K⁺ ions through the constrictions (SF, HBC and G-loop gate) of the GIRK2-Gβγ-Na⁺ system. Different hydration states are distinguished by yellow and blue colors. The incoming K⁺ ions are dehydrated through SF but partially hydrated through HBC and G-loop gate. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

(a) Secondary structure representation of two opposite subunits (chains) of GIRK2 channel with the three constrictions highlighted in red, green and purple, respectively; (b) average time duration (ns) required by K⁺ ions for permeation through SF, HBC and G-loop gate of the three wild-type systems. SF is the determinant for permeation efficiency since the $\overline{{\text{t}}_{\text{SF}}}$ is at least 4-fold of $\overline{{\text{t}}_{\text{HBC}}}$ and $\overline{{\text{t}}_{\text{G loop}}}$ in each system. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

(a) Permeation mechanism through the SF of GIRK2. Time distribution (%) of major steps and the coordination binding sites (S1-S4) are labeled. There are three translocation paths under an external voltage of 0.06 V/nm (purple arrow is shown in Path I only). The S2/S4 K⁺-bound configuration separated by water molecules takes the most simulation time, which indicates that water and K⁺ ions co-translocate through SF in a water-K⁺ coupled manner. (b) The average SF tilt angles and the standard deviations of GIRK2-Gβγ-Na⁺ (green), GIRK2-Gβγ (orange) and GIRK2-Na⁺ (red). The corresponding S1 vacancy percentage is listed below. GIRK2-Gβγ-Na⁺ shows the lowest degree of average tilt angle and fluctuation but exhibits the highest S1 occupancy percentage as a result. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Number of K⁺ ions in the SF-HBC cavity vs simulation time of (a) GIRK2-Na⁺, (b) GIRK2-Gβγ and (c) GIRK2-Gβγ-Na⁺ systems. Each black dot represents a permeation event through the HBC gate. The K⁺ permeation through an open HBC gate is only successful when the number of accumulated ions in cavity is 4 or more.

Fig. 8

Time distribution (%) of K⁺ ions in SF-HBC cavity for (a) 3-K⁺ and (b) 4-K⁺ occupancy. A total of three distributed locations of K⁺ are involved in the flux through HBC in each occupancy, and the time percentage of each location is indicated in purple, green, and yellow. With N184 as the middle boundary, the cavity can be divided into two regions, where the upper region is near E152 and the lower region is next to HBC. In order to prepare for the inward permeation, K⁺ ions in the cavity need to firstly move to the region near HBC (circled by blue dash lines). Under a 3-K⁺ pattern, K⁺ ions prefer E152 (69.8%) to the HBC gate (29.8%), which in turn hinders the preparation for permeation. In contrast, HBC is the favorite (50.2%) under 4-K⁺, which facilitates the preparation for permeation. Water molecules of hydration are removed for clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 9

(a) Pair-wise non-bonded interactions (kcal/mol) of K⁺(HBC)-residue(cavity) while four subunits of N184 carbonyl groups have unfavorable conformations for permeation through HBC. (b) Time distribution of five orientation combinations of N184 carbonyl groups under 4-K⁺ and 3-K⁺ patterns, with the favorable conformation highlighted in yellow. (c) Permeation mechanism of K⁺ through opened HBC gate. Permeation is only allowed through the 4-K⁺ pattern due to less unfavorable cation-dipole interactions (a red line on the right). Three K⁺ in the upper cavity (yellow region) are attracted by E152 to induce the formation of favorable upward orientation of N184 carbonyl groups through direct interactions. As a result, outward pulling force for permeation is decreased and the fourth K⁺(HBC) is allowed for inward transport. Water molecules of hydration are omitted for clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)