del Camino D et al. (2005), Status of the intracellular gate in the activat...

XB-ART-1162

J Gen Physiol 2005 Nov 01;1265:419-28. doi: 10.1085/jgp.200509385.

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Status of the intracellular gate in the activated-not-open state of shaker K+ channels.

del Camino D , Kanevsky M , Yellen G .

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Voltage-dependent K+ channels like Shaker use an intracellular gate to control ion flow through the pore. When the membrane voltage becomes more positive, these channels traverse a series of closed conformations before the final opening transition. Does the intracellular gate undergo conformational changes before channel opening? To answer this question we introduced cysteines into the intracellular end of the pore and studied their chemical modification in conditions favoring each of three distinct states, the open state, the resting closed state, and the activated-not-open state (the closed state adjacent to the open state). We used two independent ways to isolate the channels in the activated-not-open state. First, we used mutations in S4 (ILT; Smith-Maxwell, C.J., J.L. Ledwell, and R.W. Aldrich. 1998. J. Gen. Physiol. 111:421-439; Ledwell, J.L., and R.W. Aldrich. 1999. J. Gen. Physiol. 113:389-414) that separate the final opening step from earlier charge-movement steps. Second, we used the open channel blocker 4-aminopyridine (4-AP), which has been proposed to promote closure of the intracellular gate and thus specifically to stabilize the activated-not-open state of the channels. Supporting this proposed mechanism, we found that 4-AP enters channels only after opening, remaining trapped in closed channels, and that in the open state it competes with tetraethylammonium for binding. Using these tools, we found that in the activated-not-open state, a cysteine located at a position considered to form part of the gate (Shaker 478) showed higher reactivity than in either the open or the resting closed states. Additionally, we have found that in this activated state the intracellular gate continued to prevent access to the pore by molecules as small as Cd2+ ions. Our results suggest that the intracellular opening to the pore undergoes some rearrangements in the transition from the resting closed state to the activated-not-open state, but throughout this process the intracellular gate remains an effective barrier to the movement of potassium ions through the pore.

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Species referenced: Xenopus laevis
Genes referenced: kcna2 tbx2

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	Figure 1. 4-AP blocks Shaker only after channel opening, and it can be trapped by channel closure. Using a fast-perfusion system, 4-AP (3 mM) was applied intracellularly to an inside-out patch expressing Shaker channels. (A) Blocker was applied during a long depolarizing pulse to +60 mV and removed before returning to a holding potential of â90 mV. (B) 4-AP was perfused during a depolarizing pulse and washed out immediately after returning to a membrane potential of â90 mV. After a 2-min wash out at â90 mV, a new depolarizing step to +60 mV was applied. (C) blocker was applied for over 1 s at â90 mV and removed immediately before channel opening.
	Figure 2. TEA and 4-AP compete for blockade of locked-open Shaker channels. Shaker 476C channels were locked-open by application of 10 Î¼M total Cd2+, and soon thereafter blocker was applied by solenoid switching during a long activating pulse. Fractional blockade was determined by dividing the current in blocker (after stabilization following perfusion) by the current immediately preceding the application of blocker. Results are shown for TEA (0.4 mM) and 4-AP (15 mM), with no other blocker present. Based on these single point estimates, the Kapparent for blockade is â¼0.18 mM for TEA and â¼17.6 mM for 4-AP. The third column shows the fractional blockade by 4-AP in the constant presence of TEA (i.e., current in 4-AP plus TEA, divided by current with TEA present). The arrows show the predicted outcomes for noncompetitive (NC) and competitive (C) blockade. Noncompetitive blockade would give the same fractional blockade as seen in the absence of TEA. Competitive blockade is expected to give a Kapparent for blockade by 4-AP in the presence of TEA equal to Kapparent(4-AP)Â·(1 + [TEA]/Kapparent(TEA)), â¼56.7 mM. The predicted relative fractional blockade at 15 mM 4-AP would thus be â¼0.21. For these experiments, [K+]out was 20 mM. Data are shown as mean Â± SEM for three to four experiments.
	Figure 3. 478C becomes accessible in the activated-not-open state. (A) Gating currents for 478C-LT-W434F channels expressed in cut-open oocytes, in response to voltage steps from a holding potential of â150 mV to a pulse potential of â140 to +50 mV, in steps of 10 mV, followed by a return to â150 mV. (B) Normalized Q-V and g-V data for 478C-LT channels, obtained as described in MATERIALS AND METHODS. The square symbols indicate the mean and SEM of the voltage midpoints, and the Boltzmann g-V function has the average mean and slope from five experiments. The arrows indicate the three voltages used for chemical modification measurements. (C) A typical measurement of MTSET reaction rate for 478C-LT channels in an inside-out patch, in the presence of 4-AP. Small dots are the steady-state current measured for an activating pulse to +120 mV in the absence of 4-AP. After each large filled arrowhead, a 590-ms pulse to +110 mV was applied; after the first 80 ms, a solenoid valve delivered a 500-ms pulse of MTSET (4 Î¼M) and 4-AP (0.5 mM). The 4-AP block was complete within 25 ms, so that only a small amount of modification (<5%) occurred with unblocked channels. Small open arrowheads indicate 590 ms pulses to +110 mV with no drug application, applied to speed recovery from 4-AP application. To determine the rate constant of reaction, the points with the large arrowheads were plotted vs. cumulative modification time, and the reaction rate constant was calculated as (fitted time constant)â1 Â· [MTSET]â1. (D) Apparent second order rate constants for modification of 478C-LT channels by MTSHE and MTSET. The actual concentrations used ranged from 10 to 20 Î¼M for MTSHE, and 4 to 10 Î¼M for MTSET. Rates were determined for channels in various conditions: âclosedâ (â120 mV), âactivatedâ (â10 mV), âopenâ (+110 mV), and 4-AP bound (at +110 mV, with 0.5 mM 4-AP). The mean Â± SEM for at least three experiments is shown. For these experiments, [K+]out was 100 mM.
	Figure 4. The intracellular gate opens only when the channel finally opens, as reported by Cd2+ access to 474C. The plots show the voltage dependence for gating charge (Q-V; small symbols), channel opening (g-V), and chemical modification of 474C in 474C-ILT channels. The square symbols (mean Â± SEM, n = 3â4) indicate the second order modification rate constant inferred from experiments using total [Cd2+] in the range 4â20 Î¼M. The g-V is shown as smooth normalized Boltzmann functions with average values from 42 experiments for the midpoint and slope factor; dotted curves are Â±1 SD for these values. The bottom plot shows the same g-V and modification rate data on a logarithmic scale, indicating the close match between inferred channel open probability and modification rate.
	Figure 5. Stereogram of the Kv1.2 inner pore, illustrating the location of key pore positions in Shaker. Based on the crystal structure of Kv1.2 (Long et al., 2005a; Protein Data Bank entry 2A79). The view is from the intracellular side of the pore, after removal of the Î² subunit and T1 domain. Helices are shown as ribbons, with the S6 (inner) helices in magenta and the S4âS5 linker helices in blue. The pore-facing residues corresponding to Shaker V474 (yellow) and V478 (green) are shown as space-filling CPK models. The residues that participate in the Cd2+ lock-open bridge (Shaker V476C and H486) are shown in ball-and-stick models. Missing sidechains were restored automatically by SwissProt DeepView (http://www.expasy.org/spdbv), the H486 sidechains were manually reoriented, and the protein was displayed with a transparent Connolly surface (probe radius 1.4 A) using DS ViewerPro (Accelrys). A potassium ion with eight waters of hydration based on the high-resolution structure of KcsA (Zhou et al., 2001b; Protein Data Bank entry 1K4C) is shown in the cavity for reference.

References [+] :

Andersen, Ion movement through gramicidin A channels. Single-channel measurements at very high potentials. 1983, Pubmed