Dougherty K and Covarrubias M (2006), A dipeptidyl aminopeptidase-like protein remode...

XB-ART-35331

J Gen Physiol 2006 Dec 01;1286:745-53. doi: 10.1085/jgp.200609668.

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A dipeptidyl aminopeptidase-like protein remodels gating charge dynamics in Kv4.2 channels.

Dougherty K , Covarrubias M .

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Dipeptidyl aminopeptidase-like proteins (DPLPs) interact with Kv4 channels and thereby induce a profound remodeling of activation and inactivation gating. DPLPs are constitutive components of the neuronal Kv4 channel complex, and recent observations have suggested the critical functional role of the single transmembrane segment of these proteins (Zagha, E., A. Ozaita, S.Y. Chang, M.S. Nadal, U. Lin, M.J. Saganich, T. McCormack, K.O. Akinsanya, S.Y. Qi, and B. Rudy. 2005. J. Biol. Chem. 280:18853-18861). However, the underlying mechanism of action is unknown. We hypothesized that a unique interaction between the Kv4.2 channel and a DPLP found in brain (DPPX-S) may remodel the channel's voltage-sensing domain. To test this hypothesis, we implemented a robust experimental system to measure Kv4.2 gating currents and study gating charge dynamics in the absence and presence of DPPX-S. The results demonstrated that coexpression of Kv4.2 and DPPX-S causes a -26 mV parallel shift in the gating charge-voltage (Q-V) relationship. This shift is associated with faster outward movements of the gating charge over a broad range of relevant membrane potentials and accelerated gating charge return upon repolarization. In sharp contrast, DPPX-S had no effect on gating charge movements of the Shaker B Kv channel. We propose that DPPX-S destabilizes resting and intermediate states in the voltage-dependent activation pathway, which promotes the outward gating charge movement. The remodeling of gating charge dynamics may involve specific protein-protein interactions of the DPPX-S's transmembrane segment with the voltage-sensing and pore domains of the Kv4.2 channel. This mechanism may determine the characteristic fast operation of neuronal Kv4 channels in the subthreshold range of membrane potentials.

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Species referenced: Xenopus
Genes referenced: dpp6 kcnd2 tbx2 vsig1

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	Figure 1. CTX sensitivity of Kv4.2 wild-type and Kv4.2CTX channels expressed in Xenopus oocytes. (A and B) Outward whole-oocyte K+ currents mediated by Kv4.2CTX and Kv4.2CTX:DPPX-S channels before (control saline in the bath) and after exposure to 1 nM CTX in control saline (see Materials and methods). Currents were elicited by 400-ms step depolarizations from −100 mV to +50 mV. (C) Dose–response relationships for Kv4.2WT, Kv4.2CTX, and Kv4.2CTX:DPPX-S channels. Both Kv4.2CTX and Kv4.2CTX:DPPX-S channels are sensitive to CTX in the subnanomolar or nanomolar range. The solid line superimposed on the symbols is the best-fit Hill equation of this form: y = (A2 + (A1 − A2))/ (1 + (x/Kd)h). A1 and A2 represent the maximum and minimum, Kd represents the apparent disassociation constant, and h represents the Hill coefficient. The best-fit parameters were as follows: Kv4.2CTX, Kd = 0.83 nM and h = 1.04; and Kv4.2CTX:DPPX-S, Kd = 1.48 nM and h = 1.27 (n ≥ 3 experiments). DPPX-S does not significantly alter the CTX sensitivity of Kv.2CTX channels (P > 0.05 by one-way ANOVA). Data are expressed as mean ± SE. (D) Whole-cell (tsA201; see Materials and methods) Kv4.2CTX currents evoked by a 400-ms step depolarization to +32 mV from a holding potential of −108 mV. Note that the macroscopic inactivation of Kv4.2CTX channels is accelerated by DPPX-S. (E) Gp-V relationships of Kv4.2CTX channels expressed alone or coexpressed with DPPX-S. The solid lines are best-fit fourth-order Boltzmann functions with the following parameters: V1/2 (Kv4.2CTX) = −13.2 mV, k (Kv4.2CTX) = 23.2 mV; and V1/2 (Kv4.2CTX:DPPX-S) = −43.6 mV, k (Kv4.2CTX:DPPX) = 23.6 mV. The dashed line indicates the zero-conductance level. (F) Recovery from inactivation at −110 mV. The solid lines are the best-fit exponential functions with the following parameters: τ (Kv4.2CTX) = 186 ms; and τ (Kv4.2CTX:DPPX-S) = 116 ms. The changes induced by DPPX-S are similar to those reported by others for Kv4.2 wild type (Nadal et al., 2003; Jerng et al., 2004b). Data in E and F are expressed as mean ± SE.
	Figure 2. Ig from Kv4.2CTX and Kv4.2CTX:DPPX-S channels. (A) Ig from Kv4.2CTX channels expressed in tsA201 cells in the presence of 100 nM CTX and 105 mM intracellular CsF. From a holding potential of −153 mV, currents were elicited by a series of 12-ms voltage steps between −143 and +57 mV in 20-mV increments. (B) Ig from Kv4.2CTX:DPPX-S channels recorded under conditions identical to those used in A. Note the acceleration of the Ig-ON and Ig-OFF kinetics in the presence of DPPX-S.
	Figure 3. Charge conservation in Kv4.2CTX and Kv4.2CTX:DPPX-S channels. (A) QON-V and QOFF-V relationships from Kv4.2CTX channels. (B) QON-V and QOFF-V relationships from Kv4.2CTX:DPPX-S channels. (C) QON and QOFF values from Kv4.2CTX channels are strongly correlated with a slope of 0.97 (r = 0.98). (D) QON and QOFF values from Kv4.2CTX:DPPX-S channels are strongly correlated with a slope of 1.03 (r = 0.99).
	Figure 4. Normalized QON-V relationships from Kv4.2CTX and Kv4.2CTX:DPPX-S channels. The QON-V relationships were described by assuming a Boltzmann function (see Materials and methods) with the following best-fit parameters (Table I): V1/2 (Kv4.2 CTX) = −47 mV and z (Kv4.2 CTX) = 2.98 e0 (n = 6); and V1/2 (Kv4.2CTX:DPPX-S) = −73 mV and z (Kv4.2CTX:DPPX-S) = 3.04 e0 (n = 7). Current traces were sampled in 2-mV increments. The mean Qmax values in the absence and presence of DPPX-S were 107 ± 28 fC/pF (n = 6) and 98 ± 21 fC/pF (n = 7), respectively. Data are expressed as mean ± SE.
	Figure 5. Kv4.2CTX Ig kinetics in the absence and presence of DPPX-S. (A) Ig elicited by a voltage step to +47 mV from a holding potential of −153 mV for both Kv4.2CTX (gray traces) and Kv4.2CTX:DPPX-S (red traces) channels. Note the accelerated decay of the Kv4.2CTX:DPPX-S Ig. Continuous black lines represent the best-fit exponential decays. The dotted line is the zero-current level. (B) Time constants resulting from the exponential fits plotted against membrane potential. The continuous black lines represent the best-fit exponential (see Materials and methods). Near the midpoint voltage of the QON-V relationships (data points excluded from the exponential fit), the Ig relaxations were described by assuming the sum of two exponential terms; thus, the corresponding data points are the weighted means of the time constants. Note that at all voltages, the Kv4.2CTX:DPPX-S Ig-ON exhibits faster time constants than its Kv4.2CTX counterparts. The effective valences derived from this analysis were 1.04 ± 0.04 e0 for Kv4.2CTX (n = 6) and 0.95 ± 0.03 e0 for Kv4.2CTX:DPPX-S (n = 7). (C) OFF Ig from both Kv4.2CTX (gray traces) and Kv4.2CTX:DPPX-S (red traces) channels resulting from a repolarizing voltage step from +47 mV to −153 mV. Continuous black lines represent the best-fit sum of two exponential terms. (D) Box plot of the Ig-OFF time constants (repolarizations to −153 mV from +47 mV). The borders of the boxes represent the 25th and 75th percentiles, whereas the median and mean are represented by a closed circle and a horizontal line in the box, respectively. Error bars represent the 5th and 95th percentiles, respectively. The fractional amplitudes of the exponential terms are indicated by the numbers displayed below or above the corresponding boxes.
	Figure 6. Kinetic modeling of voltage-dependent gating in the absence and presence of DPPX-S. All simulations assume the ZHA model of Kv channel gating and were conducted as described in Materials and methods. (A) Simulated Ig evoked by 6-ms step depolarizations (top, −55 to +65 mV; bottom, −75 to +65 mV; both in 20-mV increments) to the indicated membrane potentials and assuming a holding potential of −153 mV. The top and bottom families of traces simulate Kv4.2 Ig in the absence and presence of DPPX-S, respectively. The simulation parameters are those reported in Table I of Zagotta et al. (1994), except that for the bottom family of traces, the forward rate constants (α and δ) of the two sequential transitions in each subunit of the channel were increased fivefold. The inset compares control (continuous line) and accelerated (dashed line) Ig evoked by depolarizations to +45 mV. In all plots (B–D), the simulations that assume accelerated gating are depicted by open symbols. (B) Simulated QON-V relationships. QON was simulated in 2-mV increments. The continuous lines superimposed on the simulated data symbols are best-fit Boltzmann functions (see Materials and methods) with the following parameters: V1/2 (closed symbols) = −48 mV and z (closed symbols) = 3.2 e0; and V1/2 (open symbols) = −73 mV and z (open symbols) = 3.2 e0. The ΔV1/2 = −25 mV. (C) Simulated time constant–voltage relationships. The time constants were derived by approximating the simulated Ig relaxations with an exponential function (as done for the observed Ig; see Results). The initial fast phase at low voltages and the rising phase at high voltages were excluded from this analysis. The continuous lines superimposed on the simulated data symbols are best-fit exponential functions that describe the voltage dependence of the time constants (see Materials and methods). The effective valences derived from this analysis were 1.08 e0 (closed symbols) and 1.09 (open symbols) e0. (D) Simulated G-V relationships. The plots were generated from the steady-state level of the simulated ionic currents at the indicated membrane potentials (2-mV increments). The chord conductance (G) was calculated by assuming a reversal potential of −100 mV. The continuous lines superimposed on the simulated data symbols are the best-fit fourth-order Boltzmann functions with the following parameters: V1/2 (closed symbols) = −45 mV and k (closed symbols) = 5.4 mV; and V1/2 (open symbols) = −70 mV and k (open symbols) = 5.5 mV. V1/2 is the calculated voltage at Gp/Gpmax = 0.5, and k is the slope factor. The ΔV1/2 = −25 mV.

References [+] :

Ahern, Focused electric field across the voltage sensor of potassium channels. 2005, Pubmed, Xenbase