Regulation of gating by negative charges in the cytoplasmic pore in the Kir2.1 channel

Abstract

Inward rectifier K(+) channels commonly exhibit long openings (slow gating) punctuated by rapid open-close transitions (fast gating), suggesting that two separate gates may control channel open-closed transitions. Previous studies have suggested possible gate locations at the selectivity filter and at the 'bundle crossing', where the two transmembrane segments (M1 and M2) cross near the cytoplasmic end of the pore. Wild-type Kir2.1 channels exhibit only slow gating, but mutations in the cytoplasmic pore domain at E224 and E299 have been shown to induce fast flickery gating. Since these mutations also affect polyamine affinity, we conjectured that the fast gating mechanism might affect the kinetics of polyamine block/unblock if located more intracellularly than the polyamine blocking site in the pore. Neutralization of either E224 or E299 induced fast gating and slowed both block and unblock rates by the polyamine diamine 10. The slowing of polyamine block/unblock was partly relieved by raising pH from 7.2 to 9.0, which also slowed fast gating kinetics. These findings indicate that the fast flickery gate is located intracellularly with respect to the polyamine pore-plugging site near D172, thereby excluding the selectivity filter, and implicating the bundle crossing or more intracellular site as the gate. As additional proof, fast gating induced at the selectivity filter by disrupting P loop salt bridges in WT-E138D-E138D-WT tandem had no effect on polyamine block and unblock rates. The pH sensitivity of fast gating in E224 and E299 mutants was attributed to the protonation state of H226, since the double mutant E224Q/H226K induced fast gating which was pH insensitive. Moreover, introducing a negative charge in the 224-226 region was sufficient to prevent fast gating, since the double mutant E224Q/H226E rescued wild-type Kir2.1 slow gating. These observations implicate E224 and E299 as allosteric modulators of a fast gate, located at the bundle crossing or below in Kir2.1 channels. By suppressing fast gating, these negative charges facilitate polyamine block and unblock, which may be their physiologically important role.

Figure 1. Replacement of E224 with neutral residues induces fast flickery gating kinetics and reduces single channel chord conductance

A, single channel traces recorded from inside-out patches excised from Xenopus oocytes expressing WT, E224G or E224A channels. Holding potentials (0, −80 and −160 mV) are indicated above each trace. The ratio of current variances for the open (δ²_O) and closed states (δ²_C) is indicated for each channel at −160 mV. Segments of the traces at −160 mV are expanded to compare open-channel kinetics. The arrows indicate long dwell time at a sublevel. B, all-point histograms from the single channel traces of wild-type (WT), E224G and E224A at holding potential of −160 mV. The arrows indicate substate peaks corresponding to that in A (bottom trace). C, left: Single channel i–V relation for WT (○), E224G (▵) and E224A (□) channels. Compared with the linear i–V relation in WT, mutant channels show voltage-dependent reduction of chord conductance (dotted lines show chord conductances at −160 mV). Right: normalized i–V curve from the same data.

Figure 2. Replacement of E224 with neutral residues induces intrinsic inward rectification in Kir2.1 channels and slows polyamine blocking and unblocking

A and B, representative macroscopic current traces recorded from inside-out giant patches from Xenopus oocytes expressing WT and E224A channels. Upper panel: control recordings ∼5 min after patch excision. Lower panel: in the presence of 100 μm diamine 10 (DA10). Notice the different time scales for WT and E224A. The voltage pulse protocols are illustrated at the bottom of A. Example of how block rate at −20 mV and unblock rate at −60 mV were measured are shown at the bottom of B. The data were well fitted to a single exponential function as indicated by a continuous line in each case. C, I–V relationship for WT and E224-neutralized channels, showing a blocker-independent intrinsic inward rectification in the mutant channels. D, time constants (τ) for DA10 blocking (in) at −20 mV and unblocking (out) at −60 mV obtained by a single exponential fit. ** and ##P < 0.05 compared with WT.

Figure 3. Raising pH slows fast flickery gating kinetics in E224A channels

A, single channel activity recorded at a holding potential of −100 mV. The pH of the solution was changed from 7.2 to 9.0 at the arrow under the trace. B, the expanded traces from the periods a (pH 7.2) and b (pH 9.0) in A. The ratios of current variances for the open (δ²_O) and closed states (δ²_C) are indicated for pH 7.2 and pH 9.0, respectively. C, all-point histograms from the single channel traces at pH 7.2 (left) and pH 9.0 (right). A substate observed only at pH 7.2 is as indicated by the arrow.

Figure 4. Raising pH from 7.2 to 9.0 increases both inward and outward current and weakens intrinsic inward rectification in the E224-neutralized channels

A, macroscopic current traces of WT and E224A channels at pH of 7.2 (upper panels) and 9.0 (lower panels). B, I–V relationship for WT (left) and E224A (right) at pH of 7.2 (□) and 9.0 (○), respectively. The curve at pH 7.2 for E224A is normalized (▪, 7.2N) at −80 mV to that at pH 9.0. C, the ratios of the current at +60 and −60 mV as an index of rectification intensity. ##P < 0.05 compared with pH 7.2. D, percentage increase in current at −80 mV induced by the elevation of pH from 7.2 to 9.0 in WT and mutant channels. **P < 0.05 compared with WT.

Figure 5. pH-induced gating changes affect DA10 blocking and unblocking rates in E224A channels

A, macroscopic current traces recorded from WT channels in the presence of 100 μm DA10. The elevation of pH from 7.2 (upper panels) to 9.0 (lower panels) has no effect on the rates of DA10 blocking (left) and unblocking (right). B, the same protocol for E224A channels. The elevation of pH from 7.2 to 9.0 speeds up both blocking and unblocking rates. The voltage protocols in A and B are the same with that in Fig. 1. C, summary of pH effect on DA10 unblocking rates in WT and E224A.

Figure 6. H226 is responsible for the pH sensitivity of channel gating in E224-neutralized channels

A, macroscopic current recordings from the double mutation E224Q/H224K (upper panels) and E224K/H226E (lower panels) at pH 7.2 (left) and 9.0 (right), respectively. B, normalized I–V relationships. While E244Q/H226K shows strong intrinsic inward rectification, E224Q/H226E has a WT-like linear relation. However, elevation of pH from 7.2 (squares) to 9.0 (circles) has no effect on either double mutant. C, single channel recording from E224Q/H226E exhibits a WT-like open–close kinetics without fast flickery openings.

Figure 7. H226 is responsible for the pH sensitivity of DA10 unblocking rates in the E224-neutralized channels

A and B, the elevation of pH has no effect on DA10 unblocking rate in the double mutant channels E224Q/H226K and E224Q/H226E. The voltage pulse protocol for A is illustrated at the bottom of the panel. The voltage protocol for B is the same with that shown in Fig. 2A. C, summary of pH effect on DA10 unblocking rate in the double mutant channels.

Figure 8. Structure and putative gating locations in Kir channels (based on the structure of KirBac1.1 in closed state)

Equivalent residues critical for polyamine block and fast gating in the Kir2.1 channel are indicated. The side chains of E224 at cytoplasmic pore and E138 at selectivity filter are also shown in space-filling models. In the space-filling models, blue indicates positive charges, red negative charges.

Figure 9. Fast flickery gating induced by mutation at the selectivity filter (WT-E138D–E138D-WT) does not affect DA10 block and unblock kinetics

A, single channel trace with both long and short time scales, and all-point histogram of the tandem mutant at holding potential of −120 mV. The ratio of current variances for the open (δ²_O) and closed states (δ²_C) is indicated. B, macroscopic current traces in the presence of 100 μm DA10. C, bar graphs comparing the block time constant τ_in at −20 mV and unblock time constant τ_out at −60 mV for WT-E138D–E138D-WT tandem and homologous WT channels.