Voltage-dependent neuromodulation of Na+ channels by D1-like dopamine receptors in rat hippocampal neurons

Abstract

Activation of D1-like dopamine (DA) receptors reduces peak Na+ current in acutely isolated hippocampal neurons through phosphorylation of the alpha subunit of the Na+ channel by cAMP-dependent protein kinase (PKA). Here we report that neuromodulation of Na+ currents by DA receptors via PKA is voltage-dependent in the range of -110 to -70 mV and is also sensitive to concurrent activation of protein kinase C (PKC). Depolarization enhanced the ability of D1-like DA receptors to reduce peak Na+ currents via the PKA pathway. Similar voltage-dependent modulation was observed when PKA was activated directly with the membrane-permeant PKA activator DCl-cBIMPS (cBIMPS; 20 microM), indicating that the membrane potential dependence occurs downstream of PKA. PKA activation caused only a small (-2.9 mV) shift in the voltage dependence of steady-state inactivation and had no effect on slow inactivation or on the rates of entry into the fast or slow inactivated states, suggesting that another mechanism is responsible for coupling of membrane potential changes to PKA modulation. Activation of PKC with a low concentration of the membrane-permeant diacylglycerol analog oleylacetyl glycerol also potentiated modulation by SKF 81297 or cBIMPS, and these effects were most striking at hyperpolarized membrane potentials where PKA modulation was not stimulated by membrane depolarization. Thus, activation of D1-like DA receptors causes a strong reduction in Na+ current via the PKA pathway, but it is effective primarily when it is combined with depolarization or activation of PKC. The convergence of these three distinct signaling modalities on the Na+ channel provides an intriguing mechanism for integration of information from multiple signaling pathways in the hippocampus and CNS.

Fig. 1.

Modulation of whole-cell Na⁺current by D1-like dopamine receptor activation is enhanced at depolarized holding potentials in rat hippocampal neurons.A–C, Representative current traces elicited by a test pulse to −20 mV from the indicated holding potential in control and in the presence of 5 μm SKF 81297 (smaller trace). D, Bar graph summarizing the effect of membrane depolarization on the magnitude of SKF 81297 modulation in a population of neurons (n ≥ 20 for each group). The mean of each population is indicated on the graph. Anasterisk indicates statistical significance (p ≤ 0.05; Student’s ttest).

Fig. 2.

Membrane potential-dependent modulation of Na⁺ current by cBIMPS in rat hippocampal neurons.A–C, Representative current traces elicited by a test pulse to −20 mV from the indicated holding potential in control and in the presence of 50 μm cBIMPS (smaller trace). D, Bar graph summarizing the effect of membrane depolarization on the magnitude of cBIMPS modulation in a population of neurons. An asterisk indicates statistical significance (p ≤ 0.05; Student’st test).

Fig. 3.

Membrane potential-dependent modulation of Na⁺ current by cBIMPS in tsA-201 cells transiently transfected with type IIA Na⁺ channel α subunits. A–C, Representative current traces elicited by a test pulse to 0 mV from the indicated holding potential in control and in the presence of 50 μm cBIMPS (smaller trace). D, Bar graph summarizing the effect of membrane depolarization on the magnitude of cBIMPS modulation in a population of tsA-201 cells expressing type IIA Na⁺ channel α subunits. Anasterisk indicates statistical significance (p < 0.05; Student’s ttest).

Fig. 4.

Effects of PKA phosphorylation on the voltage dependence of steady-state inactivation. A, From a holding potential of −70 mV, hippocampal neurons were depolarized to the indicated membrane potentials for 50 msec and then further depolarized to 0 mV to record peak Na⁺ currents. Steady-state fast inactivation curves are presented for control conditions (●) and in the presence of 10 μm SKF 81297 (○). B, Box plot summarizing the effects of PKA phosphorylation on the half-inactivation voltage and the slope factor. An asterisk indicates statistical significance (p ≤ 0.05; Student’s ttest). C, The peak Na⁺ current in response to test pulses to OmV is plotted as a function of time in control (●) or 50 μm cBIMPS (○) in response to depolarization of the membrane to the indicated holding potential.D, Voltage dependence of the sum of fast and slow inactivation of control and in the presence of cBIMPS derived from the data in C.

Fig. 5.

Effects of PKA phosphorylation on the rate of inactivation. A, Hippocampal neurons were depolarized to −70 mV for the indicated times and then peak currents were recorded at 0 mV. A plot of normalized peak current versus prepulse duration at −70 mV in control conditions (●) and cBIMPS (○). The solid lines are single exponential fits of the data to determine the inactivation rate constant. Note that the small increase in inactivation in the presence of cBIMPS was also observed when the pulse protocol was repeated twice in the absence of cBIMPS (▪ vs ■). B, Plot of normalized peak current versus prepulse duration at −40 mV in control and in the presence of 50 μm cBIMPS demonstrating the rate inactivation from the final closed states. C, Plot of normalized peak current versus prepulse duration at 0 mV in control and in the presence of 50 μm cBIMPS demonstrating the rate of inactivation from the open state.

Fig. 6.

Modulation by SKF 81297 and cBIMPS is enhanced by activation of PKC at a hyperpolarized holding potential in rat hippocampal neurons. A, Time course of modulation by cBIMPS in the presence and absence of OAG. The peak current is plotted as a function of time in the presence of various neuromodulators as indicated by the bars. B, C, Representative current traces elicited by a test pulse to −20 mV from a holding potential of −85 mV demonstrating modulation by SKF 81297 with or without previous activation of PKC by OAG.D, Bar graph summarizing the facilitatory effect of previous exposure to a low dose (20 μm) of OAG on the magnitude of SKF 81297 modulation in a population of neurons. Anasterisk indicates statistical significance (p ≤ 0.05; Student’s ttest).

Fig. 7.

Modulation by cBIMPS is also potentiated by PKC activation in transiently transfected tsA-201 cells expressing type IIA Na⁺ channel α subunits. A, B, Representative current traces elicited by a test pulse to 0 mV from the indicated holding potential demonstrating modulation by cBIMPS without (A) or with (B) previous activation of PKC by OAG.C, Bar graph summarizing the facilitatory effect of previous exposure to a low dose (20 μm) of OAG on the magnitude of cBIMPS modulation in a population of tsA-201 cells. Anasterisk indicates statistical significance (p ≤ 0.05; Student’s ttest).

Fig. 8.

Membrane potential-dependent enhancement of PKA modulation does not require phosphorylation by PKC. A, B, Peak current elicited by a test pulse to −20 mV from a holding potential of −70 mV plotted as a function of time in control solution and in the presence of 50 μm cBIMPS for a control cell and for a cell dialyzed with 20 μm PKCI.C, Bar graph summarizing the magnitude of PKA modulation in control and in the presence of 20 μm PKCI for a population of neurons. An asterisk indicates statistical significance (p ≤ 0.05; Student’st test).

Fig. 9.

D1/PKA modulation is voltage dependent in tsA-201 cells expressing mutant S1506A Na⁺ channel α subunits. A, B, Representative current traces elicited by a test pulse to 0 mV from the indicated holding potential in control and in the presence of 5 μm SKF 81297 (smaller trace). C, Bar graph summarizing the effect of membrane depolarization on the magnitude of SKF 81297 modulation in a population of tsA-201 cells (n ≥ 20 for each group). The mean of each population is indicated on the graph. Anasterisk indicates statistical significance (p ≤ 0.05; Student’s ttest). These results indicate that PKC phosphorylation and membrane depolarization affect PKA modulation of Na⁺ current by parallel mechanisms that both enhance the PKA-dependent reduction in peak Na⁺ current.