Allosteric effects of permeating cations on gating currents during K+ channel deactivation - PubMed (original) (raw)

Allosteric effects of permeating cations on gating currents during K+ channel deactivation

F S Chen et al. J Gen Physiol. 1997 Aug.

Abstract

K+ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K+ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K+ or Cs+, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K+ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs+ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at ECs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K+ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.

PubMed Disclaimer

Figures

Figure 1

Figure 1

(A) Uncorrected capacity transient during a 10-mV hyperpolarization from −60 mV in an HEK cell. The cell capacitance was 13.4 pF. The data were filtered at 10 kHz for presentation and are represented with a dotted line. The decay phase was fit with a single exponential function, τ = 35 μs (solid line). (B) Off-gating currents during depolarizations to between −60 and +20 mV in 20-mV steps from a holding potential of −100 mV. Capacity compensation and leak subtraction have been applied to the gating currents, but superimposed is the uncorrected capacity transient from A. The decay of each off-gating current has been fit to a monoexponential decay function with τdecay = 0.47, 0.50, 0.64, 1.24, and 1.9 ms for pulses from −60 to +20 mV.

Figure 3

Figure 3

(A) Mean normalized peak off-gating current amplitudes at −100 mV after depolarizations to between −100 and +60 mV (n = 5). Data (±SEM) obtained immediately on whole-cell access (•, n = 4) and after disappearance of any ionic current (▿, n = 5). (B) Normalized Qoff from off-gating currents as for A. Boltzmann fits gave a V0.5 of −18.2 ± 2.3 mV and z = 2.0 ± 0.1 e 0 (▿) and V0.5 of −16.7 ± 2.4 mV, z = 2.1 ± 0.2 e 0 (•). (C) τdecay of off-gating currents after prepulses to between −60 and +60 mV in cells with ionic K+ current (n = 5, •) and in cells with no ionic current (n = 4, ▿). (D) Normalized conductance-voltage (G-V, •, mean ± SEM, n = 11) curve obtained from K+ tail currents at −30 mV after 30-ms prepulses, in K+-containing intracellular and bath solutions. The Q-V (▿) curve was determined in NMG solutions (e.g., Fig. 2_A_) from the sum of on- and off-gating charge movement for each amplitude of depolarizing pulse (n = 5, ±SEM). Fits to the Boltzmann equation gave V0.5 for G and Q curves of +2.9 mV and −16.3 mV, respectively, with slope factors (k) of 9.3 and 15.3 mV (1.7 e 0).

Figure 4

Figure 4

Measurement of Kv1.5 off-gating currents with permeant Cs+. (A) On-gating and outward Cs+ currents during depolarizing prepulses from −100 to +60 mV in 20-mV steps, and off-gating currents at −100 mV (ECs); the arrow indicates the complete current decay. (Inset) Off-gating currents at ECs, −100 mV, after prepulses to +10 mV and +60 mV. τdecay was 0.48 ms on repolarization from +10 mV and 0.63 ms from +60 mV. (B) τdecay of off-gating currents after prepulses to between −60 and +60 mV in cells with no permeant ions present (data from experiments as shown in Fig. 2_A_, n = 4, ▿) and in cells with Cs+ present as the permeant cation (n = 4, ▾). (C and D) Time course of off-gating charge integrated from the off-current data in Fig. 4_A_ with Cs+ present (C) and from a cell where no permeant ions were present (D). Traces are integrated off-gating currents after prepulses in 10-mV steps, indicated by the potentials adjacent to the tracings.

Figure 5

Figure 5

Validation of off-gating current measurements made in the presence of permeant Cs+. (A) Cells were depolarized to +40 mV from −100 mV, then repolarized to test potentials between −100 and +10 mV before returning to −100 mV. (Inset) Decaying outward Cs+ tails and off-gating currents for prepulses to +40, +10, and −20 mV. Solid lines show fit to monoexponential decay functions during subsequent current waveforms recorded at −100 mV. τdecay was 0.51, 0.4, and 0.32 ms for +40-, +10-, and −20-mV prepulses, respectively. (B) K+ Ionic tail currents from Kv1.5 expressed in HEK cells. EK was −70 mV, and the cell was pulsed to +40 mV for 100 ms before repolarization to a range of potentials between −40 and −100 mV. Only decaying ionic tail currents during the repolarizing pulse are shown in B. (C) Normalized peak off-gating current (Igoff), and charge (Qoff), and values for τdecay at −100 mV after repolarizations from +40 mV (τdecay, hatched bar), from +10 mV (solid bars), and from −20 mV (empty bars). Igoff and Qoff were normalized to values obtained from immediate repolarization from +40 mV to −100 mV (n = 3, ±SEM). (D) Mean normalized Q-V relation for off-gating current (▿) and G-V for Cs+ current (•) (±SEM, n = 4). V0.5 for Qoff and G were −15.0 ± 1.3 mV (z = 1.94 ± 0.2 e 0) and −1.2 mV (k = 15.6 mV), respectively.

Figure 6

Figure 6

Envelopes of on- and off- gating currents in the absence of permeant ions with NMG as the substitute cation. (A) Gating currents during depolarizations from −100 to +60 mV and then to −100 mV to record off-gating currents. Pulse durations (frequency 0.5 Hz) were incremented 0.5 ms after the first duration of 0.25 ms to give a maximum pulse duration of 7.75 ms. Multiple sweeps of on-gating currents are superimposed in the upper left segment to show homogeneity. A decrease in off-gating current was apparent after pulse durations longer than a few ms. (B) Mean (±SEM) τdecay of off-gating currents after different prepulse durations (n = 4, •). (C) Off-gating current data when pulse duration was incremented 50 ms to give 8–358 ms durations. Currents were recorded after depolarization indicated above each segment. (D) Change in mean off-gating charge returned during the first 20 ms of repolarization after different duration depolarizations (n = 4, •).

Figure 7

Figure 7

Envelopes of on- and off-gating currents in the presence of permeant Cs+. Gating and ionic currents during depolarizations from −100 to +60 mV and then to −100 mV (ECs) to record off-gating currents. Pulse durations (frequency 0.5 Hz) were incremented 0.6 ms after the first duration of 0.45 ms to give a maximum pulse duration of 7.05 ms. Multiple sweeps of on-gating currents are superimposed in the upper left segment to show homogeneity. (B) Mean (±SEM) τdecay of off-gating currents after different prepulse durations in Cs+ (n = 6, •). For off-gating current data in C, pulse duration was incremented 50 ms to give 8–358 ms durations. Currents were recorded after depolarization indicated above each segment. (D) Change in mean off-gating charge returned during the first 20 ms of repolarization after different duration depolarizations in Cs+ (n = 8, •).

Figure 8

Figure 8

Gating currents from a nonconducting mutant of Kv1.5 channels, W472F. Permeant ions are present in the pipette and bath solutions but do not permeate through the pore as demonstrated by the lack of ionic currents present on depolarization. (A) Gating currents on depolarizations up to +60 mV from −100 mV in 20-mV steps. (Inset) Off-gating currents at −100 mV after depolarizing prepulses to −10 mV and +40 mV. τdecay was 0.53 and 1.6 ms at −10 and +40 mV, respectively. (B) Normalized Qoff from integration of off-gating currents (mean ± SEM, n = 4). V0.5 was −19.9 ± 3.6 mV and z = 2.5 ± 0.2 e 0. (C) τdecay of off-gating currents as a function of the voltage prepulse potential (mean ± SEM, n = 4). (D) Envelopes of on- and off-gating currents from Kv1.5 W472F. Protocol was as for data in Fig. 7_A_. All on-gating currents are superimposed to show homogeneity. (E) Ratio of Qoff to Qon, as a function of pulse duration at +60 mV (mean ± SEM, n = 4). (F) Slowing of τdecay of off-gating currents with increased prepulse duration (mean ± SEM, n = 4).

Figure 2

Figure 2

Voltage-dependent on- and off-gating currents of Kv1.5 in the absence and presence of permeant K+. Currents are from −100 mV for pulses to +40 mV in 20-mV steps. (A) Steady-state gating currents, 3 min after patch rupture; inset, off-gating currents at −100 mV after depolarizing prepulses to −10 and +40 mV. Solid lines indicate monoexponential fits with τ's of 0.75 ms at −10 and 2.3 ms at +40 mV. (B) Gating and ionic K+ currents immediately on achievement of whole-cell recording. Arrow indicates current decay to baseline; inset, off-gating currents at −100 mV after prepulses to −10 and +40 mV. τdecay was 0.73 ms at −10 mV and 1.11 ms at +40 mV. Data in both A and B are from the same cell. Subtraction of on-gating current waveforms before and after K+ dialysis gave faithful ionic currents on depolarization (data not shown). (C–E) On- (Qon) and off-gating (Qoff) charge time course calculated by integration of on-gating current in A (C), and off-gating currents in A (D) and B (E). Note that for all data where gating charge was measured by integration of off-gating current transients, integration times were sufficient to allow currents to relax to baseline. This was usually complete within 25 ms.

Figure 9

Figure 9

Schematic diagram of two pathways by which allo-steric modulation of channel gating may occur during short depolarizations. Channels may traverse the path outlined by the dashed box in the absence of permeating cations or in a nonconducting mutant, W472F. Immediately preceding the open state (O) is a late closed state (C) in the activation pathway from which channels proceed slowly to O and rapidly to an absorbing inactivated state (I) from which return is slow. With permeating K+ or Cs+, a state where cations are bound within the pore (OK) exists that shows rapid transitions to and from C (open arrow). Access to the inactivated state is not significant during short depolarizations when permeant ions are present. The scheme with permeating ions is then defined by the solid box.

Similar articles

Cited by

References

    1. Armstrong CM, Bezanilla F. Currents related to movement of the gating particles of the sodium channels. Nature (Lond) 1973;242:459–461. - PubMed
    1. Baukrowitz, T., and G. Yellen. 1995. Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms. Neuron .15:951–960. - PubMed
    1. Baukrowitz T, Yellen G. Use-dependent blockers and exit rate of the last ion from the multi-ion pore of a K+channel. Science (Wash DC) 1996;271:653–656. - PubMed
    1. Bezanilla F, Perozo E, Papazian DM, Stefani E. Molecular basis of gating charge immobilization in Shaker potassium channels. Science (Wash DC) 1991;254:679–683. - PubMed
    1. Bezanilla F, Perozo E, Stefani E. Gating of Shaker K+channels. II. The components of gating currents and a model of channel activation. Biophys J. 1994;66:1011–1021. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources