Modulation of the Shaker K(+) channel gating kinetics by the S3-S4 linker - PubMed (original) (raw)
Modulation of the Shaker K(+) channel gating kinetics by the S3-S4 linker
C Gonzalez et al. J Gen Physiol. 2000 Feb.
Abstract
In Shaker K(+) channels depolarization displaces outwardly the positively charged residues of the S4 segment. The amount of this displacement is unknown, but large movements of the S4 segment should be constrained by the length and flexibility of the S3-S4 linker. To investigate the role of the S3-S4 linker in the ShakerH4Delta(6-46) (ShakerDelta) K(+) channel activation, we constructed S3-S4 linker deletion mutants. Using macropatches of Xenopus oocytes, we tested three constructs: a deletion mutant with no linker (0 aa linker), a mutant containing a linker 5 amino acids in length, and a 10 amino acid linker mutant. Each of the three mutants tested yielded robust K(+) currents. The half-activation voltage was shifted to the right along the voltage axis, and the shift was +45 mV in the case of the 0 aa linker channel. In the 0 aa linker, mutant deactivation kinetics were sixfold slower than in ShakerDelta. The apparent number of gating charges was 12.6+/-0.6 e(o) in ShakerDelta, 12.7+/-0.5 in 10 aa linker, and 12.3+/-0.9 in 5 aa linker channels, but it was only 5.6+/-0.3 e(o) in the 0 aa linker mutant channel. The maximum probability of opening (P(o)(max)) as measured using noise analysis was not altered by the linker deletions. Activation kinetics were most affected by linker deletions; at 0 mV, the 5 and 0 aa linker channels' activation time constants were 89x and 45x slower than that of the ShakerDelta K(+) channel, respectively. The initial lag of ionic currents when the prepulse was varied from -130 to -60 mV was 0.5, 14, and 2 ms for the 10, 5, and 0 aa linker mutant channels, respectively. These results suggest that: (a) the S4 segment moves only a short distance during activation since an S3-S4 linker consisting of only 5 amino acid residues allows for the total charge displacement to occur, and (b) the length of the S3-S4 linker plays an important role in setting ShakerDelta channel activation and deactivation kinetics.
Figures
Scheme S1
Scheme S2
Scheme S3
Figure 1
Mutations of the Shaker S3–S4 linker region. The wild-type Shaker sequence (top) is compared with the mutations studied here. The S3–S4 linker was defined according to Wallner et al. 1996. The asparagine-arginine-serine sequence in the 10 aa linker channel corresponds to the S3–S4 linker region present in the human calcium-activated K+ channel (h_Slo_; Wallner et al. 1996).
Figure 2
Deletions in the S3–S4 linker slow down activation and deactivation kinetics in _Shaker_Δ. Macroscopic currents for _Shaker_Δ (Α), and 10 (B), 5 (C), and 0 (D) aa S3–S4 linker mutants. Currents were recorded from cell-attached macropatches from oocytes expressing the different channels. Currents were elicited by voltage steps from −60 to 120 mV in 5-mV increments, followed by a step to −60 mV. The holding voltage was −100 mV.
Figure 3
Deletions in the S3–S4 linker shift the G-V curves toward depolarizing voltages. Voltage dependence of activation of wild-type _Shaker_Δ and S3–S4 linker mutants. The conductance–voltage curves were obtained using tail current measurements. Each point is the average of determinations on 10–20 separate patches. Solid lines were drawn using parameters in Table and for the WT and 10 aa linker mutant, or for the 5 and 0 aa linker mutants. Dashed lines drawn for the WT and 10 aa linker mutant are the best fit to a fourth power Boltzmann function.
Figure 4
Activation kinetics become slower by deletions in the S3–S4 linker. (A) Channel activation was characterized by a fit of the time course of the current elicited by depolarization from a holding potential of −100 mV to and exponential function with a time constant τa and a time delay d (). Arrows in the inset show d for the 5 and 0 aa linker mutants. The fit started near the time at which current had reached ∼30–50% of the maximum (see inset). Arrows in the inset show d for the 5 and 0 aa linker mutants. The solid lines are fits to the data using an equation of the form: τa = A exp(−_Z_V/RT), where τa is the activation time constant, A is a constant, Z defines the voltage dependence of τa, and V is the voltage. Parameters A were 2.45 ± 0.74, 4.45 ± 0.25, 217 ± 65, and 111 ± 22 ms for the WT, and 10, 5, and 0 aa linker mutants, respectively. Parameters Z were 0.95 ± 0.35, 1.30 ± 0.03, 0.71 ± 0.08, and 0.53 ± 0.04 for the WT, and 10, 5, and 0 aa linker mutants, respectively. (B) Plots of the time delay d as a function of voltage. Lines were drawn using an equation of the form: d = A exp(−_Z_V/RT). Parameters A were 3.1 ± 1.1, 146 ± 53, and 103 ± 48 ms for the 10, 5, and 0 aa linker mutants, respectively. Parameters Z were 1.11 ± 0.10, 0.70 ± 0.05, and 1.68 ± 0.06 for the 10, 5, and 0 aa linker mutants, respectively.
Figure 5
The time constants of current deactivation were obtained by fitting the tail currents after a 250-ms activating pulse to 15 mV as indicated in the pulse protocol shown in the inset. Time course was fitted with a single exponential. Solid lines are fit to the data using τd = C exp(−_Z_V/RT), where τd is the deactivation time constant, C is a constant, and Ζ defines the voltage dependence of τd. The values of Z were: 0.53 ± 0.9, 0.8 ± 0.08, 0.58 ± 0.1, and 0.69 ± 0.08 for the WT, and 10, 5, and 0 aa linker mutants, respectively.
Figure 6
Kinetics of early transitions of the activation pathway using the Cole-Moore protocol. (A) Family of macroscopic ionic currents from 5 aa linker mutant channels elicited in response to the variable hyperpolarized prepulses shown in the pulse protocol followed by a 50-mV test pulse and a postpulse of −90 mV. The holding voltage was −90 mV. A P/−4 subtraction protocol was used. Data was filtered at 5 kHz and digitized every 100 μs. (B) Delay or time shift due to the hyperpolarized prepulses for the linker mutant channels calculated from current records was measured by displacing the current records along the time axis until the best superposition was obtained. Lines in these plots were drawn by eye.
Figure 7
Limiting slope analysis in wild-type _Shaker_Δ and the S3–S4 linker mutant channels. (A–D) Semilogarithmic plot of the G vs. V relationship of the different mutants. The solid line indicates the fitting of the low probability data in order to determine the limiting value of z. (E–H) z vs. V plot of the corresponding mutants indicated above the A–D graphs. The solid line indicates the asymptotic value of z at very hyperpolarizing voltages obtained by the derivative with respect to the voltage of the monoexponential fit of the low probability data.
Figure 8
Variance analysis for three deletion mutants. (A,D, and G) Mean current traces obtained from 256 traces recorded with the patch technique from a holding potential of −100 mV to a test pulse potential of 120 mV. (B, E, and H) Time course of the variance. (C,F, and I) Variance versus current fitted to the function σ_2_= _i_I(t) − I(t)2/N (solid line). The mean maximum open probability (P o max) among the different mutants was 0.75.
Figure 9
Model of the structure of all six segments of the Shaker K channel, which accounts for the observations described in the present study (Cha et al. 1999; Bezanilla 2000). (A) Two subunits facing each other across the pore are shown. This allows the observation of the back face of the left subunit and the front face of the right subunit. Only the open configuration of the channel is shown. In this model, depolarization rotates the S4 segment in 180°, and charges that were facing the intracellular solution by being in the crevice formed by S1 and S5 are displaced towards the extracellular solution remaining in a water-filled crevice formed by segments S2 and S3. This requires an S4 movement of no more than a few Angstroms and explains why a 5 aa linker allows for the movement of all the channel gating charges. (B) When the whole S3–S4 linker is removed, the S3 and S4 segments are closer together, decreasing the width of the crevice formed by the S2 and S3 segments. This has the effect of widening the region where the electric field falls, with the consequent decrease in _z_δ.
Similar articles
- Periodic perturbations in Shaker K+ channel gating kinetics by deletions in the S3-S4 linker.
Gonzalez C, Rosenman E, Bezanilla F, Alvarez O, Latorre R. Gonzalez C, et al. Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9617-23. doi: 10.1073/pnas.171306298. Epub 2001 Aug 7. Proc Natl Acad Sci U S A. 2001. PMID: 11493701 Free PMC article. - Deletion of the S3-S4 linker in the Shaker potassium channel reveals two quenching groups near the outside of S4.
Sørensen JB, Cha A, Latorre R, Rosenman E, Bezanilla F. Sørensen JB, et al. J Gen Physiol. 2000 Feb;115(2):209-22. doi: 10.1085/jgp.115.2.209. J Gen Physiol. 2000. PMID: 10653897 Free PMC article. - Role of the S3-S4 linker in Shaker potassium channel activation.
Mathur R, Zheng J, Yan Y, Sigworth FJ. Mathur R, et al. J Gen Physiol. 1997 Feb;109(2):191-9. doi: 10.1085/jgp.109.2.191. J Gen Physiol. 1997. PMID: 9041448 Free PMC article. - Gating of voltage-dependent potassium channels.
Fedida D, Hesketh JC. Fedida D, et al. Prog Biophys Mol Biol. 2001;75(3):165-99. doi: 10.1016/s0079-6107(01)00006-2. Prog Biophys Mol Biol. 2001. PMID: 11376798 Review. - Voltage sensor movement in the hERG K+ channel.
Piper DR, Sanguinetti MC, Tristani-Firouzi M. Piper DR, et al. Novartis Found Symp. 2005;266:46-52; discussion 52-6, 95-9. doi: 10.1002/047002142x.ch5. Novartis Found Symp. 2005. PMID: 16050261 Review.
Cited by
- N-terminal region is responsible for mHv1 channel activity in MDSCs.
Peña-Pichicoi A, Fernández M, Navarro-Quezada N, Alvear-Arias JJ, Carrillo CA, Carmona EM, Garate J, Lopez-Rodriguez AM, Neely A, Hernández-Ochoa EO, González C. Peña-Pichicoi A, et al. Front Pharmacol. 2023 Oct 17;14:1265130. doi: 10.3389/fphar.2023.1265130. eCollection 2023. Front Pharmacol. 2023. PMID: 37915407 Free PMC article. - Binding of RPR260243 at the intracellular side of the hERG1 channel pore domain slows closure of the helix bundle crossing gate.
Zangerl-Plessl EM, Wu W, Sanguinetti MC, Stary-Weinzinger A. Zangerl-Plessl EM, et al. Front Mol Biosci. 2023 Feb 23;10:1137368. doi: 10.3389/fmolb.2023.1137368. eCollection 2023. Front Mol Biosci. 2023. PMID: 36911523 Free PMC article. - Na+ and K+ channels: history and structure.
Armstrong CM, Hollingworth S. Armstrong CM, et al. Biophys J. 2021 Mar 2;120(5):756-763. doi: 10.1016/j.bpj.2021.01.013. Epub 2021 Jan 21. Biophys J. 2021. PMID: 33484711 Free PMC article. Review. - Structural Dynamics of the Paddle Motif Loop in the Activated Conformation of KvAP Voltage Sensor.
Das A, Chatterjee S, Raghuraman H. Das A, et al. Biophys J. 2020 Feb 25;118(4):873-884. doi: 10.1016/j.bpj.2019.08.017. Epub 2019 Aug 22. Biophys J. 2020. PMID: 31547975 Free PMC article. - NMR Structural Analysis of Isolated Shaker Voltage-Sensing Domain in LPPG Micelles.
Chen H, Pan J, Gandhi DM, Dockendorff C, Cui Q, Chanda B, Henzler-Wildman KA. Chen H, et al. Biophys J. 2019 Jul 23;117(2):388-398. doi: 10.1016/j.bpj.2019.06.020. Epub 2019 Jun 26. Biophys J. 2019. PMID: 31301804 Free PMC article.
References
- Aggarwal S.K., MacKinnon R. Contribution of the S4 segment to gating charge in the Shaker K+ channel. Neuron. 1996;16:1169–1177. - PubMed
- Almers W. Gating currents and charge movements in excitable membranes. Rev. Physiol. Biochem. Pharmacol. 1978;82:96–190. - PubMed
- Andersen O., Koeppe R.E., II. Molecular determinants of channel function. Physiol. Rev. 1992;72:S89–S158. - PubMed
- Baker O.S., Larsson H.P., Mannuzzu L.M., Isacoff E.Y. Three transmembrane conformation and sequence-dependent displacement of the S4 domain in Shaker K+ channel gating. Neuron. 1998;20:1283–1294. - PubMed
- Bezanilla F. The voltage sensor in voltage-dependent ion channels. Physiol. Rev. 2000;80:555–592. - PubMed