Initial steps in the opening of a Shaker potassium channel

Abstract

The structural model of a K(V) (K(+)-selective, voltage-gated) channel in the open state is known (Protein Data Bank ID code 2R9R). Each subunit of the channel has four negatively charged residues distributed in the transmembrane segments S1, S2, and S3 that bind to and facilitate the movement within the membrane of the positively charged, voltage-sensing residues of S4. When extrapolated to the closed state, the two outermost negatively charged residues are exposed to extracellular fluid and not bound to S4 residues, all of which have theoretically been driven inward by voltage. If this closed state model is correct, these residues are available to bind external cations. We examined the effects of La(3+) on voltage-gated Shaker K(+) channels. Addition of the trivalent cation La(3+) (50 μM) extracellularly markedly prolongs the lag that precedes channel opening and slows the subsequent rise of K(+) current (I(K)) at all voltages. Decay kinetics of I(K) at negative voltages are unaltered. Gating current (I(g)) recorded from a nonconducting mutant shows that La(3+) reduces the initial amplitude of I(g) nearly twofold. We postulate that, in the resting state, La(3+) binds to the unoccupied, outermost negative residues, hindering outward S4 motion, thus increasing the lag on activation and slowing the rise of I(K). In the activated state, La(3+) is displaced by outward movement of arginine residues in S4; La(3+), therefore, is not present to affect channel closing. The results give strong support to the closed state model of the K(V) channel and a clear explanation of the effect of multivalent cations on cellular excitability.

Fig. 1.

The resting and activated conformations of the K_V1.2–2.1 voltage-sensing domain. (A) Experimentally determined activated conformation (Protein Data Bank ID code 2R9R). Note that R1 in Kv1.2–2.1 is glutamine, whereas R1 in Shaker is arginine. (B) Hypothetical resting/closed conformation. The yellow sphere represents the Ca²⁺ in the hypothetical binding area. The images were created using Mac PyMol v0.9. The residues are labeled using the numbering in K_V1.2–2.1. Modified from Long et al. (11).

Fig. 2.

Slowing of I_K activation by extracellular La³⁺. (A) Representative I_K traces without (black) and with (blue) 50 μM La³⁺. I_K was recorded from ShB∆6–46 T449V, and La³⁺ was applied to the extracellular side. Currents were elicited by 6.5-ms depolarization pulses to the voltages indicated from the holding potential of −80 mV. The traces without La³⁺ are the average of a set taken before application of La³⁺ and another closely similar set after washing it away. (B) Peak outward I-V curves without (black) and with (blue) 50 μM La³⁺. The data points are from the experiment in A and are connected by straight lines for clarity.

Fig. 3.

Changes in I_g elicited by depolarization by extracellular La³⁺ without altering the total charge movement (Q_g) in ShB∆6–46 W434F. (A) Representative I_g without (black) and with (blue) 50 μM La³⁺ elicited by pulses from −80 mV to 40, 20, 0, and −20 mV (from Top to Bottom) at 0.5 Hz. The traces without La³⁺ are the average of a set taken before application of La³⁺ and another closely similar set after washing it away. (B) Voltage dependence of Q_g estimated from integrals of I_g from the traces in A. Black, without La ³⁺; blue, with La³⁺.

Fig. 4.

La³⁺ has no effect on I_K deactivation after repolarization. (A) Representative I_K without (black) and with (blue) 50 μM La³⁺ in the extracellular medium from a cell expressing ShB∆6–46 T449V. Currents were elicited by 12-ms pulses to 20 mV and then, −80 mV. (B) The currents shown in A are scaled by a factor of 1.29 to facilitate comparison of the kinetics. Channels were activated by a 1-ms pulse to 20 mV, and I_K tails were recorded on returning V_m to −80 mV.

Fig. 5.

I_g kinetics on repolarization. Representative I_g values from ShB∆6–46 W434F at −80 mV after 14-ms activating pulses to the voltages indicated are shown. The black and blue traces show the results obtained without and with La³⁺ (50 μM), respectively. In addition, the results obtained after a pulse to −30 mV were scaled and are shown in Inset.