Mechanism of Cd2+ coordination during slow inactivation in potassium channels

Abstract

In K+ channels, rearrangements of the pore outer vestibule have been associated with C-type inactivation gating. Paradoxically, the crystal structure of Open/C-type inactivated KcsA suggests these movements to be modest in magnitude. In this study, we show that under physiological conditions, the KcsA outer vestibule undergoes relatively large dynamic rearrangements upon inactivation. External Cd2+ enhances the rate of C-type inactivation in an cysteine mutant (Y82C) via metal-bridge formation. This effect is not present in a non-inactivating mutant (E71A/Y82C). Tandem dimer and tandem tetramer constructs of equivalent cysteine mutants in KcsA and Shaker K+ channels demonstrate that these Cd2+ metal bridges are formed only between adjacent subunits. This is well supported by molecular dynamics simulations. Based on the crystal structure of Cd2+ -bound Y82C-KcsA in the closed state, together with electron paramagnetic resonance distance measurements in the KcsA outer vestibule, we suggest that subunits must dynamically come in close proximity as the channels undergo inactivation.

Figure 1. Cd²⁺ Increases the Rate of Inactivation in Y82C-KcsA

Normalized macroscopic currents of Y82C mutant of KcsA, reconstituted in asolectin liposomes, at a depolarizing potential (+150 mV) for concentrations of Cd²⁺ ranging from 0 to 1 mM (A-D); in the background of non-inactivating mutant E71A in the presence (red) and absence (black) of 100 μM Cd²⁺ (F). Macroscopic responses of Y82C-KcsA under various conditions were elicited by pH jumps from 8.0 to 4.0 using a rapid solution exchanger in the presence of 200 mM KCl and with a membrane potential held at +150 mV. Inactivation time constant (τ_i) as a function of various Cd²⁺ concentrations is shown in (E), n > 5.

Figure 2. Mobility Differences of Spin-labeled Y82C in Reconstituted WT and E71A Channels

Effect of opening the lower gate on the mobility of spin-labeled outer-vestibule residue Y82C in reconstituted wild type (top) and non-inactivating mutant E71A (bottom) backgrounds for the closed (pH 7, red) and open (pH4, black) states of KcsA, as determined by CW-EPR.

Figure 3. Cd²⁺Metal Bridge is Formed between Adjacent Subunits

Normalized currents of (A) TD-Y82C (diagonal cysteines) and (B) TT-Y82C-KcsA (adjacent cysteines), reconstituted in asolectin liposomes, in the absence (black) and presence of 100 μM Cd²⁺ (red). Macroscopic responses of tandem constructs were elicited by pH jumps from 8.0 to 4.0 using a rapid solution exchanger in the presence of 200 mM KCl and with a membrane potential held at +150 mV, n > 5. A schematic representation of the position of cysteines in the TD (tandem dimer) and TT (tandem tetramer) constructs of Y82C-KcsA is shown.

Figure 4. Stability of Cd²⁺Coordinated States

(A) Change in S-Cd²⁺ distance during brute force molecular dynamics simulations (350 ps) in Y82C-KcsA for Cd²⁺ coordinated initially between adjacent (top) and diagonal (bottom) configurations. Snapshots of initial and final configurations are shown in top view. (B) Change in free energy for linearly pulling Cd²⁺ from a coordination state between adjacent subunits to a coordination state between diagonal subunits. Snapshots of configurations along the path are shown. Cadmium is represented by a grey sphere, and side chains of Y82C for the relevant two subunits are shown in sticks. See Experimental Procedures and text for details, and Supplementary Movies online.

Figure 5. Crystal structure of the Cd²⁺-bound Y82C-KcsA at 2.4 Å resolution

Shown are the 2F_o-F_c electron density map of the side view for diagonally symmetric subunits [left; sigma for protein (blue mesh) = 2.0, sigma for potassium (magenta mesh) = 2.5, sigma for cadmium (green mesh) = 3.2] and top view (right) of the tetramer structure depicting the individually bound Cd²⁺ (green spheres) to Y82C cysteines. The polypeptide chain is shown as sticks. The S_γ-S_γ distances between cysteines corresponding to diagonal and adjacent subunits are shown (black arrows). See Experimental Procedures and text for details.

Figure 6. Subunits come closer dynamically during inactivation gating

(A) CW-EPR distance determination between spin labels bound to diagonally placed Y82C in TD KcsA in the closed (pH 7, top) and open (pH 4, bottom) forms of KcsA. Distance measurements were made in proteoliposomes at 130 K in the presence of liq. N₂ using <50 μW incident power to avoid saturation of the signal. The full and under label EPR spectra are shown in the left, and the probability of distance distributions with the mean distance is shown on right. A cartoon depicting the closed and opened tandem dimer channels as well as the position of spinlabeled Y82C in the outer-vestibule is shown. (B) The crystal structure of spinlabel-bound Y82C-KcsA tetramer at 2.5 Å is shown (left, top view) along with the 2F_o-F_c electron density map of the side view for diagonally symmetric subunits [right; sigma for protein (blue mesh) = 2.0, sigma for potassium (magenta mesh) = 2.5] depicting the spinlabel bound to Y82C cysteines. The polypeptide chain is shown as sticks. The distance between the N-O groups of the spinlabel (12.7 Å) from two diagonally symmetric subunits is shown (black arrow). See Experimental Procedures and text for details.

Figure 7. Cd²⁺ metal bridge formation between adjacent subunits is highly favorable in Shaker K⁺ channels

Two-electrode voltage clamp measurements for various ShΔ T449C constructs expressed in oocytes corresponding to channels in which (A) cysteine is present in only one subunit (TTCT); (B) cysteines in diagonal subunits (TC); (C) cyseines in adjacent subunits (TTCC) and (D) cysteines in all subunits (T449C monomer). The traces represent the current recordings during 8 s depolarizations to +40 mV in the absence (red) and presence of 100 μM Cd²⁺ (blue) and subsequent wash (green). Inset shows the cartoon representation of channels highlighting the subunit containing T449C mutations (blue). The holding potential was -90 mV and the time interval between pulses is 30 s. n > 5. A star represents the point where the amplitude of current is measured for each pulse in all cases. The effect of Cd²⁺ on inactivation kinetics in ShΔ T449C constructs is shown in (E) for TTCT (black squares); TC (blue squares); TTCC (majenta squares) and T449C monomer (red squares). See Experimental Procedures for details.

Figure 8. Mechanism of stabilization of inactivated state by Cd²⁺

Addition of Cd²⁺ binds to individual cysteines at the outer-vestibule in the closed state. When the channel is opened, Cd²⁺ rearranges to form favorable metal bridges with cysteines of adjacent subunits due to structural rearrangements of the outer-vestibule. This possibly stabilizes the C-type inactivated state and results in decrease in the K⁺ current amplitude and increase in the rate of inactivation in K⁺ channels. Our model suggests that formation of the Cd²⁺ metal bridge is absolutely essential to affect the rate of C-type inactivation, and the formation of metal bridge is favored only in the inactivated state. See Discussion for details.