Ternary Kv4.2 channels recapitulate voltage-dependent inactivation kinetics of A-type K+ channels in cerebellar granule neurons

Abstract

Kv4 channels mediate most of the somatodendritic subthreshold operating A-type current (I(SA)) in neurons. This current plays essential roles in the regulation of spike timing, repetitive firing, dendritic integration and plasticity. Neuronal Kv4 channels are thought to be ternary complexes of Kv4 pore-forming subunits and two types of accessory proteins, Kv channel interacting proteins (KChIPs) and the dipeptidyl-peptidase-like proteins (DPPLs) DPPX (DPP6) and DPP10. In heterologous cells, ternary Kv4 channels exhibit inactivation that slows down with increasing depolarization. Here, we compared the voltage dependence of the inactivation rate of channels expressed in heterologous mammalian cells by Kv4.2 proteins with that of channels containing Kv4.2 and KChIP1, Kv4.2 and DPPX-S, or Kv4.2, KChIP1 and DPPX-S, and found that the relation between inactivation rate and membrane potential is distinct for these four conditions. Moreover, recordings from native neurons showed that the inactivation kinetics of the I(SA) in cerebellar granule neurons has voltage dependence that is remarkably similar to that of ternary Kv4 channels containing KChIP1 and DPPX-S proteins in heterologous cells. The fact that this complex and unique behaviour (among A-type K(+) currents) is observed in both the native current and the current expressed in heterologous cells by the ternary complex containing Kv4, DPPX and KChIP proteins supports the hypothesis that somatically recorded native Kv4 channels in neurons include both types of accessory protein. Furthermore, quantitative global kinetic modelling showed that preferential closed-state inactivation and a weakly voltage-dependent opening step can explain the slowing of the inactivation rate with increasing depolarization. Therefore, it is likely that preferential closed-state inactivation is the physiological mechanism that regulates the activity of both ternary Kv4 channel complexes and native I(SA)-mediating channels.

Figure 2. Immunolocalization of Kv4.2 and Kv4.3 proteins in the mouse cerebellar cortex

Immunostaining of sagittal sections of the mouse cerebellum with antibodies to Kv4.2 and Kv4.3. The figure shows the Cy3 fluorescence signal. Kv4.2 is expressed predominantly in the granule cell layer (GCL); with prominent staining in anterior, but weak staining in posterior lobules. Kv4.3 is strongly expressed in the molecular layer (MoL), where the dendrites of Purkinje cells are brightly stained. In the granule cell layer (GCL) Kv4.3 immunostaining is prominent in posterior lobules and extremely weak in anterior lobules. Insets show higher magnification images of the GCL illustrating Kv4.2 and Kv4.3 labelling in the periphery of granule cell somata and in the glomeruli containing the dendritic processes of the granule cells. Scale bar: 800 μm; insets: 30 μm.

Figure 6. Kinetic modelling of Kv4.2 currents

A, kinetic scheme for Kv4.2 channel gating based on the models proposed by Kaulin et al. (2008). To obtain the model's best-fit for each condition, the analysis was constrained simultaneously by all traces in the average family of currents, the average steady-state inactivation curve (not shown) and the average recovery from inactivation (Fig. 1F and Methods). The open-state inactivation pathway is enclosed in a box (dashed lines). B and C, families of observed (B) and best-fit (C) currents elicited by step depolarizations from −99 to +41 mV in 20 mV increments from a holding potential of −149 mV. The number of averaged families of observed currents is indicated in the plots. The best-fit parameters are shown in Table 1.

Figure 1. Modulation of the inactivation kinetics of Kv4.2 channels by associated proteins KChIP1 and DPPX-S

A–D, whole-cell K⁺ currents from tsA-201 cells transfected with Kv4.2 (A), Kv4.2 and DPPX-S (B), Kv4.2 and KChIP1 (C) or Kv4.2 + KChIP1 and DPPX-S (D), evoked by step depolarizations from −109 to +61 mV in 10 mV increments from a holding potential of −149 mV. Note that DPPX accelerates and KChIP1 slows down inactivation kinetics. E, voltage dependence of the rate of inactivation of Kv4.2 channels. Plots of the half-inactivation time (t_1/2) against membrane potential from the currents recorded in tsA-201 cells expressing Kv4.2 alone (•), Kv4.2 and DPPX-S (○), Kv4.2 and KChIP1 (▵) and Kv4.2 plus KChIP1 and DPPX-S (grey triangles). Shown are means s.e.m. (n = 4, 4, 10 and 10, respectively). Note that, for currents recorded in the presence of KChIP1 and KChIP1 plus DPPX-S, the rate of inactivation increases with strong depolarizations. However, DPPX accelerates the rate of inactivation between −60 and −10 mV, increasing the voltage range over which the t_1/2 increases with progressive depolarization. F, recoveries from inactivation of Kv4.2 alone (•), Kv4.2 and DPPX-S (○), Kv4.2 and KChIP1 (▵) and Kv4.2 plus KChIP1 and DPPX-S (grey triangles); the recovery voltages were −115, −128, −140 and −140 mV, respectively. These voltages were adjusted to account for shifts in the voltage dependence of steady-state inactivation (not shown), and therefore are 40 mV more negative than the midpoint voltage of the corresponding steady-state inactivation curve. The curves shown are means s.e.m. (n = 3, 3, 8 and 9, respectively) and the lines are best-fit single exponentials with the following time constants: 165, 40, 96 and 45 ms, for Kv4.2, Kv4.2:DPPX-S, Kv4.2:KChIP-1 and Kv4.2:KChIP-1:DPPX-S, respectively.

Figure 3. K⁺ currents in cerebellar granule cells

Whole-cell ionic currents recorded in a representative cerebellar granule cell from an anterior (A and C) or a posterior (B and D) lobule, before (A and B) or after (C and D) the application of 5 mm TEA. Currents were recorded in the presence of 500 nm TTX and were elicited by step depolarizations from −90 to +60 mV in 10 mV increments from a holding potential of −130 mV.

Figure 4. Inactivation kinetics of I_SA in cerebellar granule cells

A and B, whole-cell K⁺ currents recorded in a representative cerebellar granule cell from an anterior (A) or a posterior (B) lobule recorded in the presence of 5 mm TEA. Shown are the currents elicited by step depolarizations from −90 to +60 mV in 10 mV increments, preceded by a prepulse to −100 mV from a holding potential of −130 mV (upper traces) or a prepulse to −40 mV (middle traces). The lower traces show the transient currents (I_SA) obtained by subtracting the currents recorded during depolarizing steps preceded by a prepulse to −40 mV from the currents obtained during depolarizing steps preceded by a prepulse to −100 mV. C and D, half-inactivation time (t_1/2) as a function of membrane potential of the I_SA obtained in cerebellar granule cells from anterior (C) and posterior (D) lobules. Shown are means s.e.m. (n = 17 for anterior and n = 6 for posterior lobules). E, normalized peak conductance–voltage relations (G/G_max) and steady-state inactivation curves (I/I_max) for granule cells recorded in anterior (squares) and posterior (circles) cerebellar lobules. Peak conductance (G) was calculated as G = I_p/(V_m−V_eq), where I_p, V_m and V_eq are the peak current, the test potential and the K⁺ equilibrium potential, respectively. Shown are means s.e.m. (n = 21 for anterior and n = 6 for posterior lobules). The continuous lines across the data points are the best-fits to Boltzmann functions with a V_1/2=−74 and −82 mV a slope factor k = 6.9 and 5.9 mV for the inactivation curves in anterior and posterior lobules, respectively; and a V_1/2=−15 and −21 mV a slope factor k = 22 and 19 mV for the conductance–voltage curves in anterior and posterior lobules, respectively. F, recovery from inactivation of I_SA at −130 mV in cells recorded from anterior (squares) and posterior (circles) lobules. Shown are means s.e.m. (n = 11 for anterior and n = 6 for posterior lobules). The traces are single exponential fits through the data.

Figure 5. The voltage dependence of the rate of inactivation of the I_SA in cerebellar granule cells is a property of a homogeneous population of channels

A and B, representative records of the currents used to determine inactivation rates. Shown are the currents recorded during a test pulse to −30 mV (A) or +20 mV (B) preceded by a prepulse to +10 mV applied 10, 50 or 500 ms before the test pulse. Between the prepulse and the test pulse the cell was held at −110 mV. C and D, half-inactivation time (t_1/2) as a function of membrane potential of the I_SA obtained in cerebellar granule cells from anterior (C) and posterior (D) lobules during test pulses to the indicated voltages after a 10 ms (diamonds), 50 ms (triangles), or 500 ms (circles) recovery period at −110 mV from the inactivation produced by a prepulse to +10 mV. Black squares are the t_1/2 for the currents recorded during identical test pulses in the absence of a prepulse. Shown are means s.e.m. (n = 4 for anterior and n = 3 for posterior lobules).

Figure 7. Kinetic modelling of Kv4.2 inactivation

A, plots of half-inactivation time against voltage (t_1/2–voltage relation) for simulated currents mediated by Kv4.2 channels with different subunit compositions. The t_1/2 was obtained from the best-fit currents shown in Fig. 6C. B, effect of voltage dependence of the opening equilibrium on the t_1/2–voltage relation. In one case (•), the rate constants ɛ and φ are voltage-dependent (Fig. 6A; Table 1), and in the other (continuous line), these rate constants are assumed to be voltage-independent. All other best-fit parameters are kept as depicted in Table 1. C, best-fit recoveries from inactivation of Kv4.2 channels with different subunit compositions. Continuous lines are best single exponential fits with the following time constants: 156 ms (Kv4.2); 47 ms (Kv4.2:DPPX-S); 99 ms (Kv4.2:DPPX-S); and 49 ms (Ternary). These values are in excellent agreement with the experimental observations (Fig. 1 legend).