Intermediate conductances during deactivation of heteromultimeric Shaker potassium channels - PubMed (original) (raw)

Intermediate conductances during deactivation of heteromultimeric Shaker potassium channels

J Zheng et al. J Gen Physiol. 1998 Oct.

Abstract

A previous study of the T442S mutant Shaker channel revealed activation-coupled subconductance levels that apparently represent kinetic intermediates in channel activation (Zheng, J., and F.J. Sigworth. 1997. J. Gen. Physiol. 110:101-117). We have now extended the study to heteromultimeric channels consisting of various numbers of mutant subunits as well as channels without mutant subunits, all in the background of a chimeric Shaker channel having increased conductance. It has been found that activation-coupled sublevels exist in all these channel types, and are traversed in at least 80% of all deactivation time courses. In symmetric K+ solutions, the currents in the two sublevels have a linear voltage dependence, being 23-44% and 54-70% of the fully open conductance. Sublevels in different channel types share similar voltage dependence of the mean lifetime and similar ion selectivity properties. However, the mean lifetime of each current level depends approximately geometrically on the number of mutant subunits in the channel, becoming shorter in channels having fewer mutant subunits. Each mutant subunit appears to stabilize all of the conducting states by approximately 0.5 kcal/mol. Consistent with previous results in the mutant channel, sublevels in channels with two or no mutant subunits also showed ion selectivities that differ from that of the fully open level, having relatively higher K+ than Rb+ conductances. A model is presented in which Shaker channels have two coupled activation gates, one associated with the selectivity filter and a second associated with the S6 helix bundle.

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Figures

Figure 1

Figure 1

Coexpression of WT and mutant cRNAs produces channels with six distinct phenotypes. (A) Representative single channel currents, activated by a depolarizing pulse from a −100 mV holding potential to −60 mV, followed by a hyperpolarizing pulse to −140 mV. Dotted lines indicate baselines; currents were filtered at 1 kHz. (B) Normalized tail currents at −120 mV were assembled from idealized single channel recordings of each channel type. Given in each panel is the time that the current decays to its half peak amplitude. (C) Amplitude histogram of each channel type, measured at −60 mV. The fully open and closed levels are seen as the major peaks; the small intermediate-current components seen in the lower histograms arise from poorly resolved closures and from occasional long-lived dwells in sublevels not related to activation. (D) A cartoon illustrating possible combinations of WT and mutant subunits, represented by ○ and •, respectively, corresponding to the channel phenotypes. M, mutant subunit; W, WT subunit.

Figure 2

Figure 2

Distributions of channel types. (A) Cumulative histograms of single channel amplitudes observed in patches from oocytes coinjected with WT and mutant cRNAs at the various ratios (W:M) indicated. Current amplitudes, measured at −60 mV, were taken from fits of the amplitude histograms as shown in Fig. 1_C_ with a mixture of Gaussian functions. Dotted lines indicate the mean of each group, whose value is given in the bottom panel. (B) Histograms showing the number of observed channels belonging to each group. The data were fitted to a binomial distribution (solid lines) and the probability ratio that generated the maximum likelihood is given as P m/P w in each panel.

Figure 3

Figure 3

Two types of amplitude histograms from tail currents. (A) A representative current trace from the M4 channel. The holding potential was −100 mV. The dashed line indicates zero current level; data were filtered at 1 kHz. Two thresholds (dotted lines) were used to detect the times when current crossed 10 and 90% of the fully open current level during deactivation at −100 mV. (B) Tail histogram, made by accumulating all the data points in the tail current time course from 148 sweeps, scaled to units of milliseconds per picoampere. Superimposed are Gaussian functions fitted to each peak (dotted curves) and their sum (solid curve). The peak position of each Gaussian gives an estimate of each current amplitude, and the area under each Gaussian gives an estimate of the mean dwell time of that state. This histogram included sweeps in which the channel failed to deactivate during the 200-ms recording period; thus, the sublevel components are artifactually reduced in area. (C) Transition histogram, made by accumulating those data points that fell between the final crossing of the 90% threshold and the subsequent crossing of the 10% threshold. This histogram shows more clearly the dwells in sublevels. (D) Simulated current steps of unit amplitude after Gaussian filtering (1 kHz, solid curve ; 5 kHz, dotted curve) show finite dwell times in the transition range. (E) Scaled amplitude histograms from the time courses in_D_. Notice that the apparent dwell times from a current step at 1 kHz are much shorter than those obtained from the tail transitions in C.

Figure 4

Figure 4

Sublevels in each channel type. (A) Representative single-channel deactivation time courses at −100 mV. The repolarization to −100 mV preceded the beginning of each trace, which shows only the vicinity of the recording where the deactivation transition occurred. Dotted lines indicate baselines. Data were filtered at 1 kHz except for W4, which was filtered at 5 kHz. Two sweeps from a M2W2Lchannel and two sweeps from a M2W2H channel are shown to demonstrate two types of closing transitions, one through sub2and sub1, the other through sub2′. Two W4 sweeps are also presented (with expanded time scale as shown). The left sweep shows a transition with an obvious dwell in sub2, the right sweep has no apparent dwells in sublevels. (B) Tail histograms at −100 mV, from 148 (M4), 223 (M3W), 298 (M2W2L), 165 (M2W2H), 40 (MW3), and 412 (W4) sweeps, respectively. (C) The corresponding transition histograms, obtained as in Fig. 3_C_. Thresholds defining the transitions were 10 and 90% except in the case of W4, where the thresholds were set at 15 and 85% of the fully open current amplitude.

Figure 5

Figure 5

Voltage dependence of the mean lifetimes in each current level. Mean dwell times of the open (A), sub2 (B), and sub1 (C) states were estimated from Gaussian fits to transition histograms as in Fig. 4. Mean dwell times for the M4 channel were measured by threshold analysis (Zheng and Sigworth, 1997); the histogram analysis of M4 currents yielded similar results. The standard deviations are also shown for each channel type at −100 mV. The numbers of channels measured for each channel type are: W4, n = 2; MW3, n = 5; M2W2L,n = 4; M2W2H, n = 3; MW3, n = 2; W4, n = 2. The dotted curve in each panel represents the prediction from the kinetic scheme for the M4channel given by Zheng and Sigworth (1997). The brief lifetimes of sublevels in MW3 and W4 channels precluded their measurement at voltages below −120 and above −80 mV.

Figure 6

Figure 6

Closing transitions at −100 mV are characterized by the time the final transition spent between 90 and 10% of the current amplitude. Superimposed on the histograms are results of maximum-likelihood fits to each measured value assuming that the closures are either instantaneous transitions from fully open current to zero current level (whose measured dwells are approximated by a Gaussian distribution, Eq. 5) or transitions through one (for M2W2L, MW3, and W4) or two (for M4 and M3W) sublevels, whose measured dwells are approximated by one or two exponential distributions convolved with a Gaussian probability density function with the same variance (Eq. 6). The time constant of the exponential functions and the fraction A 0 of the instantaneous component are: M4, τ1 = 12.1 ms, τ2 = 3.2 ms, 8%; M3W, τ1 = 6.9 ms, τ2 = 0.9 ms, 4%; M2W2L, τ = 3.6 ms, 17%; MW3, τ = 1.1 ms, 18%; W4, τ = 0.26 ms, 20%.

Figure 7

Figure 7

Correlation of dwell times in sublevels. Activation transitions at −60 mV were filtered at 1 kHz and dwells in each sublevel were measured by a pair of thresholds that bracketed it. Only those sweeps showing a unidirectional progression of conductance levels were counted. Each plotted point represents the apparent dwell times in sub1 and sub2, except in the right-hand M2W2L graph where dwells in sub2′ are plotted against the total time spent in the two larger sublevels. In each graph, the dotted curves represent the locus of points obtained from simulations in which one or the other sublevel is skipped during activation. In all channel types, there were many transitions having nonzero dwells in both current levels, except for the M2W2L channel, for which only 2 of 167 deactivations showed a long dwell in both the sub2–sub1 level and the sub2′ level. The M2W2H channels behaved similarly to the M2W2Lchannels (data not shown).

Figure 8

Figure 8

Current amplitudes with Rb+ ion as the charge carrier. (A) Representative tail currents of M2W2L (top) and W4 (bottom) channels recorded at −100 mV with 140 mM Rb+ ion in the pipette solution. (B) Tail histograms of M2W2L and W4 channels. (C) Transition histograms accumulated from the final closing transitions. The two peaks in the M2W2L histogram have areas of 5.0 and 3.1 ms, respectively. The peak in the W4 histogram has an area of 0.6 ms. (D) Ratio of Rb+ to K+ current amplitudes in the fully open level and in sublevels, measured at −100 mV.

Scheme I

Scheme I

Scheme II

Scheme II

Scheme III

Scheme III

Figure 9

Figure 9

Mean lifetime of each current level depends on the number of mutant subunits. The number of channels measured is given in parentheses. Values from M2W2H channels are presented as dotted symbols to distinguish them from those of M2W2L channels; for these two channel types, the sub2′ dwells are also plotted (▵). Solid lines are fits to the lifetimes at −100 mV of an exponential function in which n is the number of mutant subunits as expected from Eq. 9. The k values are 0.90 (Open), 1.01 (Sub2), and 1.04 (Sub1). The dotted line represents the predicted lifetime of the open state assuming that each subunit undergoes independent transitions (Eq. 8). In this case, the transition rates (βm and βw) of mutant and WT subunits were taken from the mean open time (t) of the corresponding homomultimeric channels as βx = 1/(4_t_ x).

Figure 10

Figure 10

Hypothetical free energy profiles for M4 (solid curve) and W4 channels (dotted curve), drawn with gating charge movement as the reaction coordinate. (A) Energy profile during channel activation at −60 mV. (B) Energy profile during deactivation at −120 mV. The free energy change for each transition and the associated charge were calculated from the kinetic model of M4channels (Zheng and Sigworth, 1997). The free energy differences between M4 and W4 channels were calculated from the mean lifetimes of each state. The free energy at transition states was taken to be the same between M4 and W4 for simplicity, and because the entry into conducting states (the first latency) is essentially unaffected by the mutation (Zheng and Sigworth, 1997).

Figure 11

Figure 11

Two activation gating schemes. (A) The scheme of Zagotta et al. (b) is shown, in which a single subunit undergoes two voltage-dependent conformational transitions (inset) to reach a permissive state (○ and •). Subunit transitions occur independently, with the exception of a slowed transition from the open state 14 to state 13. The scheme has been modified to identify sublevels sub1 and sub2with states in which two or three subunits are in the permissive state. (B) The scheme is modified to include a distinct transition (dotted line) that follows activation of the subunits. This allosteric transition is assumed to switch between nonconducting states and open states having different conductances. The equilibrium constants θs1, θs2, and θo are greatly increased by the T442S mutation. In both schemes, the conducting states are represented by • and ▴.

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