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. 2004 Jan 7;24(1):35-42.
doi: 10.1523/JNEUROSCI.3807-03.2004.

Production of resurgent current in NaV1.6-null Purkinje neurons by slowing sodium channel inactivation with beta-pompilidotoxin

Tina M Grieco  1 , Indira M Raman
Affiliations

Production of resurgent current in NaV1.6-null Purkinje neurons by slowing sodium channel inactivation with beta-pompilidotoxin

Tina M Grieco et al. J Neurosci. .

Abstract

Voltage-gated tetrodotoxin-sensitive sodium channels of Purkinje neurons produce "resurgent" current with repolarization, which results from relief of an open-channel block that terminates current flow at positive potentials. The associated recovery of sodium channels from inactivation is thought to facilitate the rapid firing patterns characteristic of Purkinje neurons. Resurgent current appears to depend primarily on NaV1.6 alpha subunits, because it is greatly reduced in "med" mutant mice that lack NaV1.6. To identify factors that regulate the susceptibility of alpha subunits to open-channel block, we voltage clamped wild-type and med Purkinje neurons before and after slowing conventional inactivation with beta-pompilidotoxin (beta-PMTX). beta-PMTX increased resurgent current in wild-type neurons and induced resurgent current in med neurons. In med cells, the resurgent component of beta-PMTX-modified sodium currents could be selectively abolished by application of intracellular alkaline phosphatase, suggesting that, like in NaV1.6-expressing cells, the open-channel block of NaV1.1 and NaV1.2 subunits is regulated by constitutive phosphorylation. These results indicate that the endogenous blocker exists independently of NaV1.6 expression, and conventional inactivation regulates resurgent current by controlling the extent of open-channel block. In Purkinje cells, therefore, the relatively slow conventional inactivation kinetics of NaV1.6 appear well adapted to carry resurgent current. Nevertheless, NaV1.6 is not unique in its susceptibility to open-channel block, because under appropriate conditions, the non-NaV1.6 subunits can produce robust resurgent currents.

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Figures

Figure 1.
Figure 1.
β-PMTX slows conventional sodium channel inactivation and increases steady-state sodium current in three cell types. A, Cells were held at -90 mV. Left panels, Transient currents evoked by steps from -90 to 0 mV in control (black traces) and β-PMTX (10 μm; gray traces). Dotted lines on all traces indicate 0 pA. Right panels, Time constants of inactivation (τdecay) in wild-type Purkinje (WT PKJ, top), wild-type CA3 (WT CA3, middle), and med Purkinje (med PKJ, bottom) neurons. Asterisks in all figures indicate p < 0.05. B, Steady-state sodium current (Iss) in control conditions (open symbols) and in β-PMTX (filled symbols) for WT PKJ (circles), WT CA3 (triangles), and med PKJ (inverted triangles) cells. Mean Iss at each potential was normalized to peak sodium current at 0 mV (Ipeak (0 mV)) in each cell.
Figure 2.
Figure 2.
Effects of β-PMTX are voltage dependent. A, The percentage of steady-state relative to peak sodium current (100 * Iss/Ipeak) in wild-type Purkinje (WT PKJ), wild-type CA3 (WT CA3), and med Purkinje (med PKJ) cells in control and β-PMTX-containing solutions, at potentials between -20 and 10 mV. Symbols are as in Figure 1B. Dashed lines indicate linear fits to the data. B, Plot of the slope of Iss/Ipeak fitted in A for all classes of cells. Dotted line indicates zero slope. Ctrl, Control.
Figure 3.
Figure 3.
β-PMTX increases the amplitude of resurgent sodium current in wild-type Purkinje neurons. A, Currents elicited by step depolarizations from -90 to +30 mV, followed by step repolarizations to -30 mV in control (black traces) and β-PMTX (red traces) for wild-type Purkinje (WT PKJ, top) and wild-type CA3 (WT CA3, bottom) neurons. Transient currents are off scale (tildes). Calibration bars refer to both sets of traces. Vertical dashed lines indicate region expanded in B. B, Currents elicited by 100 msec repolarizing steps to potentials between -70 and -10 mV in 20 mV increments, as indicated by arrows. Calibration bars refer to both sets of traces. For the illustrated cells, the transient current at -30 mV in β-PMTX was 67% (wild-type Purkinje) and 108% (CA3) of control. C, Mean current-voltage relationship for currents recorded in control (black symbols) and β-PMTX (red symbols) for wild-type Purkinje (top) and wild-type CA3 neurons (bottom). Peak current was measured as the maximal current occurring >300 μsec after the repolarization, which excluded the extremely fast tail that was evident in a few wild-type Purkinje cells, but included the wild-type CA3 tail current.
Figure 4.
Figure 4.
β-PMTX induces resurgent sodium current in med Purkinje neurons. A, Currents elicited by step depolarizations from -90 to +30 mV, followed by step repolarizations to -30 mV in control (black traces) and β-PMTX (red traces) for med Purkinje neurons (med PKJ). B, Currents elicited by 100 msec repolarizing steps to potentials between -70 and -10 mV in 20 mV increments, as labeled. Transient currents are off scale (tildes). For the illustrated cell, the med Purkinje transient current at -30 mV in β-PMTX was 112% of control. C, The I-V relationship of repolarization-evoked currents in med Purkinje neurons recorded in control (black squares) and β-PMTX (red squares). Wild-type Purkinje (WT PKJ) data from Figure 3C is illustrated in gray for comparison.
Figure 5.
Figure 5.
Sensitivity of resurgent current to dephosphorylation in both wild-type and med Purkinje inside-out patches. A, Currents elicited in inside-out patches by depolarizations from -90 to 0 mV (left) and by repolarizations from +30 to -30 mV (right). Data from one wild-type Purkinje (WT PKJ, top traces) and two med Purkinje (med PKJ, middle and bottom traces) patches before (black lines) and after (gray lines) exposure to alkaline phosphatase (3 mg/ml). Transient currents (left) were normalized to reveal differences in inactivation kinetics. Calibration bar in top left panel applies to all left panels. B, Inside-out patches from wild-type Purkinje (WT PKJ, top) and med Purkinje (med PKJ, bottom) neurons with 10 μm β-PMTX included in the extracellular (pipette) solution before (left) and after (right) exposure to phosphatase. Traces are averaged data from six wild-type or four med patches. Calibration bars apply to both left and right panels.
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