Jerng HH et al. (1999), Inactivation gating of Kv4 potassium channels: ...

XB-ART-13085

J Gen Physiol 1999 May 01;1135:641-60.

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Inactivation gating of Kv4 potassium channels: molecular interactions involving the inner vestibule of the pore.

Jerng HH , Shahidullah M , Covarrubias M .

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Kv4 channels represent the main class of brain A-type K+ channels that operate in the subthreshold range of membrane potentials (Serodio, P., E. Vega-Saenz de Miera, and B. Rudy. 1996. J. Neurophysiol. 75:2174- 2179), and their function depends critically on inactivation gating. A previous study suggested that the cytoplasmic NH2- and COOH-terminal domains of Kv4.1 channels act in concert to determine the fast phase of the complex time course of macroscopic inactivation (Jerng, H.H., and M. Covarrubias. 1997. Biophys. J. 72:163-174). To investigate the structural basis of slow inactivation gating of these channels, we examined internal residues that may affect the mutually exclusive relationship between inactivation and closed-state blockade by 4-aminopyridine (4-AP) (Campbell, D.L., Y. Qu, R.L. Rasmussen, and H.C. Strauss. 1993. J. Gen. Physiol. 101:603-626; Shieh, C.-C., and G.E. Kirsch. 1994. Biophys. J. 67:2316-2325). A double mutation V[404,406]I in the distal section of the S6 region of the protein drastically slowed channel inactivation and deactivation, and significantly reduced the blockade by 4-AP. In addition, recovery from inactivation was slightly faster, but the pore properties were not significantly affected. Consistent with a more stable open state and disrupted closed state inactivation, V[404,406]I also caused hyperpolarizing and depolarizing shifts of the peak conductance-voltage curve ( approximately 5 mV) and the prepulse inactivation curve (>10 mV), respectively. By contrast, the analogous mutations (V[556,558]I) in a K+ channel that undergoes N- and C-type inactivation (Kv1.4) did not affect macroscopic inactivation but dramatically slowed deactivation and recovery from inactivation, and eliminated open-channel blockade by 4-AP. Mutation of a Kv4-specific residue in the S4-S5 loop (C322S) of Kv4.1 also altered gating and 4-AP sensitivity in a manner that closely resembles the effects of V[404, 406]I. However, this mutant did not exhibit disrupted closed state inactivation. A kinetic model that assumes coupling between channel closing and inactivation at depolarized membrane potentials accounts for the results. We propose that components of the pore's internal vestibule control both closing and inactivation in Kv4 K+ channels.

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Species referenced: Xenopus
Genes referenced: kcna4 kcnd1 tbx2

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	Figure 2. Macroscopic currents recorded from Xenopus oocytes expressing Kv4.1 K+ channels. (A) Wild-type macropatch outward currents evoked by 900-ms step depolarizations from a holding potential of −100 mV to test potentials from −80 to +60 mV in 10-mV increments (the displayed currents are the average of three consecutive runs). The interpulse interval was 5 s. (B) V[404,406]I outward currents evoked as described in A. Recordings in A and B were obtained in the cell-attached configuration of the patch-clamp method (materials and methods). (C) Comparison of normalized currents at +60 mV from A and B. (D) Normalized whole-oocyte outward currents evoked by a 9-s step depolarization to +50 mV from a holding potential of −100 mV. (E) The rising phase of the macroscopic currents evoked as described in C. For comparison, the currents are shown normalized. These currents were low-pass filtered at 2.5 kHz and digitized at <100 μs/point (the displayed currents are the average of 15 consecutive runs). (F) The time course of recovery from inactivation at −100 mV. This experiment recorded whole-oocyte currents elicited by a double pulse protocol. From a holding potential of −100 mV, a step depolarization to +40 mV evoked the control current and allowed inactivation to occur (500 ms for wild-type channels and 9 s for mutant channels). After a variable interval (interpulse interval [IPI]) at the same holding potential, a second 250-ms pulse was delivered to test for the recovery of the current. The interepisode interval was 5 s. The peak value of the current evoked by the second pulse is divided by that of the current evoked by the first pulse. The graph shows this ratio as a function of the IPI. The open symbols and bars represent the means ± SD of three experiments. To compare the same interpulse intervals, the graph shows a single experiment from wild type (for additional results see Table I). These data were described assuming an exponential rise (solid lines). The best-fit estimates of the time constants were 343 and 265 ms for wild-type and mutant channels, respectively.
	Figure 3. Activation and inactivation properties of macroscopic Kv4.1 currents. (A) The peak G/V relation. The experiments were conducted as described in Fig. 2 (A and B). The peak chord conductance (G) was calculated according to this relation: G = Ip/ (Vc − Vr), where Ip is the peak current, Vc is the command voltage (from −80 to +60 mV), and Vr is the reversal potential of the current (−95 mV). The peak G/V relation was described assuming a fourth order Boltzmann distribution (Zagotta et al., 1994). For comparison and display, G is divided by Gmax (the estimated maximal conductance) and plotted against the membrane potential (command voltage). The symbols and bars represent the means ± SD of nine and seven patches from oocytes expressing wild-type and mutant channels, respectively. The solid lines represent the best-fit fourth-order Boltzmann distributions. The best-fit parameters were: V0.5 = −47 mV and slope factor = 22 mV/e-fold, for the wild-type channels; and V0.5 = −53 mV and slope factor = 15 mV/e-fold, for the mutant channels. (B) Prepulse inactivation curves. The prepulse inactivation protocol consisted of a sequence of two pulses: a 10-s prepulse that was varied between −120 and −10 mV in 10-mV increments and a 250-ms test pulse to +40 mV. The holding potential during the interepisode interval (5 s) was −100 mV. The peak of the current evoked by the test pulse was plotted against the prepulse voltage. This relation was described assuming a Boltzmann distribution. For comparison and display, Ip is divided by Imax (the maximal control current) and plotted against the prepulse potential. The symbols and bars represent the means ± SD of five and seven patches from oocytes expressing wild-type and mutant channels, respectively. The solid lines represent the best-fit Boltzmann distributions. The best-fit parameters were: V0.5 = −71 mV and slope factor = 6 mV/e-fold, for the wild-type channels; and V0.5 = −59 mV and slope factor = 3.5 mV/e-fold, for the mutant channels. (C) The time course of prepulse inactivation of wild-type channels at −70 mV. The pulse sequence is shown in the inset. From a holding potential of −100 mV a control 500-ms pulse to +40 mV was delivered to obtain the control current. This pulse was followed by a 5-s interval at −100 mV (to recover from inactivation that developed during the first pulse). Then, the membrane was held for a variable interval at a prepulse potential (∼V0.5 of prepulse inactivation) before delivering a 500-ms test pulse to +40 mV. The interepisode interval was 5 s. (D) Comparison of the time courses of prepulse inactivation from wild-type and V[404,406]I channels at their corresponding midpoints voltages of prepulse inactivation. Graph displays the relation between the normalized current (I/Imax) and the duration of the prepulse. Symbols and bars represent the means ± SD (wild-type: n = 3; V[404,406]I: n = 4). The data were described assuming an exponential decay (solid lines). The best-fit estimates of the time constants were 2.3 and 6.7 s for wild-type and mutant channels, respectively.
	Figure 4. Deactivation kinetics of Kv4.1 K+ currents. (A) Wild-type tail currents. After a pulse to +50 mV to activate the current, the membrane was repolarized to membrane potentials between −140 and 0 mV in 10-mV increments. The interepisode interval was 5 s. (B) V[404,406]I tail currents. Currents evoked as described for A. (C) Scaled and superimposed tail currents from A and B at −140 mV. The thin solid lines through the traces are the best fit exponential (wild type) or biexponential curves (V[404,406]I). The estimated time constants were 1.2 ms for wild-type, and 5 (19%) and 41 (81%) ms for V[404,406]I. The numbers in parenthesis are the relative weights of the exponential terms. (D) Time constants of the tail current relaxations as a function of membrane potential. The dotted symbols represent the fast time constants and the open symbols represent the slow time constants.
	Figure 5. Kv4.1 single channel currents. (A and C) Consecutive single channel traces evoked by a step depolarization to +50 mV from a holding potential of −100 mV. The dotted line represents the closed level, and the arrows indicate the beginning and end of the depolarizing pulse. Note rapid flickering and unresolved openings in the wild-type traces, and a more stable open state in the mutant traces. A clear sublevel is resolved in the first mutant trace. The excessively prolonged latency to the first opening in the last mutant trace was not consistently observed (see results). The corresponding ensemble average currents (n = 64 traces) are displayed in the lower part of A and C. All recordings were obtained in the cell- attached configuration. (B and D) The single channel current–voltage relations of Kv4.1 channels. Two methods were used to measure the unitary conductance (γ): (a) by ramping the membrane potential between −100 and +70 mV or (b) by determining the mean unitary current (from amplitude histograms) at various membrane potentials (+30, +40, +50, +60, and +70 mV). Because of rapid flickering and the presence of apparent subconductance levels in the recordings from wild-type channels, only well-resolved full openings were considered in this analysis (note that the mean unitary amplitudes appear slightly underestimated). The solid lines across the currents and the symbols are linear regression fits to the mean values of the unitary currents. Symbols and bars represent the mean ± SD (n = 3 and 4 patches in B and D, respectively).
	Figure 6. Macroscopic properties of C322S channels. (A) Whole-oocyte outward currents evoked by a 900-ms step depolarization to +50 mV from a holding potential of −100 mV. (B) Macropatch outward currents evoked by a 9.9-s step depolarization to +50 mV from a holding potential of −100 mV (C322S only). Mutant and wild-type currents in A and B are shown normalized. The inset shows the rising phase of the currents at +50 mV. The dotted lines in A and B represent the zero current level. (C) Time constants of tail current relaxations. The experiments were conducted and analyzed as described in Fig. 4. The mutant current relaxations were best described assuming the sum of two exponential terms (open and dotted symbols). The wild-type values are replotted from Fig. 4. Symbols and bars represent the mean ± SD (C322S, n = 6). (D) Time course of prepulse inactivation. This experiment was conducted and analyzed as described in Fig. 3. For C322S channels, the prepulse potential was −68 mV. The time course corresponding to C322S channels (open symbols) was described assuming an exponential decay (τ = 2.6 s). The data for wild type are replotted from Fig. 3. Symbols and bars represent the mean ± SD (C322S, n = 3).
	Figure 7. Macroscopic inactivation of Kv4.1 channels in the presence and absence of the cytoplasmic NH2- and COOH-terminal domains. Whole-oocyte currents were elicited by the indicated pulse protocol. All traces are shown normalized. The biophysical properties of the double deletion mutant (ΔN71/ΔC158) were characterized in a previous study (Jerng and Covarrubias, 1997).
	Figure 8. Macroscopic Kv1.4 K+ currents expressed in Xenopus oocytes. (A) Whole-oocyte currents evoked by 900-ms step depolarizations from a holding potential of −100 mV to test pulses from −80 to +50 mV in 10-mV increments. The interepisode interval in all experiments was 10 and 45 s for wild-type and mutant channels, respectively. (Right) A comparison of normalized wild-type and mutant currents at +50 mV. (B) The effect of membrane potential on the time constant of the tail current relaxations. The experiments were conducted and analyzed as described in Fig. 4. The main difference was that the interpulse interval for the recording of mutant currents was prolonged (30 s). Symbols and bars represent the mean ± SD (wild-type, n = 5; mutant, n = 5). (C) The time course of recovery from inactivation at −100 mV. The experiment was conducted and analyzed as described in Fig. 2. The time constants of recovery from inactivation (solid lines) were 2 and 12 s for wild-type and mutant channels, respectively.
	Scheme IA.
	Scheme IB.
	Scheme II.
	Scheme III.
	Figure 9. Simulated K+ currents according to Scheme III. (A) Outward currents generated by assuming a 1-s voltage-step from −100 to +50 mV. The parameters of the simulation were as shown in Table III. The key for the calculated traces is shown on the right-hand side of B. To calculate the “Mutant-1” trace, only the closing rate k−1 was reduced (39.5 s−1). To calculate the “Mutant-2” trace, the parameters were adjusted to approximate V[404,406]I currents (Table III). The decay of the calculated wild-type (WT) current is well described assuming a sum of three exponential terms: τ1 = 17 ms (0.2), τ2 = 60 ms (0.4), and τ3 = 250 ms (0.4); the sustained level was <0.01 (relative weight of the exponential term in parentheses). These values are consistent with the results of the analysis of Kv4.1 inactivation (Table I). The degree of inactivation of the simulated Mutant-2 current [I(450 ms)/Ipeak] is 0.8, which is 10-fold higher than the value calculated from the WT current. These values are also in agreement with observations (see text). (B) Outward currents generated by assuming a 10-s voltage step from −100 to +50 mV. (C) The rising phase of the calculated outward currents shown in A. The T50% of the WT and Mutant-2 traces are 1.8 and 3.2 ms, respectively, and the corresponding activation delays were 1.2 and 1.5 ms (see text). Similar values were measured from the observed currents. (D) Inward tail currents generated by assuming a protocol similar to that described in Fig. 4 (Vtail = −140 mV). The WT and Mutant-2 currents can also be described assuming exponential relaxations with time constants that agree with the observations (Fig. 4). Dotted lines represent the zero current level.

References [+] :

Aiyar, Topology of the pore-region of a K+ channel revealed by the NMR-derived structures of scorpion toxins. 1995, Pubmed