Participation of Kv1 channels in control of membrane excitability and burst generation in mesencephalic V neurons

Abstract

The function and biophysical properties of low threshold Kv1 current in control of membrane resonance, subthreshold oscillations, and bursting in mesencephalic V neurons (Mes V) were examined in rat brain stem slices (P8-P12) using whole cell current and voltage patch-clamp methods. alpha-dendrotoxin application, a toxin with high specificity for Kv1.1, 1.2, and 1.6 channels, showed the presence of a low-threshold K(+) current that activated rapidly around -50 mV and was relatively noninactivating over a 1-s period and had a V(1/2)max of -36.2 mV. Other toxins, specific for individual channels containing either Kv 1.1, 1.2, or 1.3 alpha-subunits, were applied individually, or in combination, and showed that Kv1 channels are heteromeric, composed of combinations of subunits. In current-clamp mode, toxin application transformed the high-frequency resonant properties of the membrane into a low-pass filter and concomitantly reduced the frequency of the subthreshold membrane oscillations. During this period, rhythmical bursting was transformed into low-frequency tonic discharge. Interestingly, in a subset of neurons that did not show bursting, low doses of alpha-dendrotoxin (alpha-DTX) sufficient to block 50% of the low threshold Kv1 channels induced bursting and increased the resonant peak impedance and subthreshold oscillations, which was replicated with computer simulation. This suggests that a critical balance between inward and outward currents is necessary for bursting. This was replicated with computer simulation. Single cell RT-PCR and immunohistochemical methods confirmed the presence of Kv1.1, 1.2, and 1.6 alpha-subunits in Mes V neurons. These data indicate that low threshold Kv1 channels are responsible for membrane resonance, contribute to subthreshold oscillations, and are critical for burst generation.

FIG. 1.

Membrane resonance, subthreshold oscillations, and burst generation during maintained membrane depolarization in mesencephalic V (Mes V) neurons. A: maintained positive current injection induces rhythmical burst discharges that emerge from the underlying subthreshold oscillations. B: boxed segment in A at an expanded amplitude and time scale. Note the sinusoidal, waxing, and waning subthreshold oscillations preceding burst onset (action potentials are truncated). C: sinusoidal constant current stimulus of increasing frequency shows the presence of a frequency-dependent subthreshold voltage response indicative of membrane resonance. D: impedance-frequency relationship shows that the membrane behaves like a band-pass filter and thus has resonant properties (holding potential −55 mV).

FIG. 2.

The effects of α-dendrotoxin (α-DTX) on Mes V neuron discharge properties. A: α-DTX (100 nM) transformed burst discharge into tonic discharge. B: summary plot of the frequency-spike interval relationship before and during toxin application. Although spike discharge is tonic and of lower frequency after toxin application, only the 1st 40 spike intervals after stimulus onset are shown for comparison with spiking during burst discharge (control; n = 9 neurons, SE bars shown). The x-axis represents the approximate number of spike intervals for a typical burst. C: action potentials elicited by 1-s, 300-pA current step in the same neuron before and after addition of 100 nM α-DTX to the external solution. Stimulus was subthreshold for burst generation. Under control conditions, 2 spikes were elicited by a 300-pA current pulse. After α-DTX, the neuron showed tonic discharge. D: summary composite plot of number of actions potentials evoked as a function of stimulus intensity. Toxin application clearly reduced the threshold to elicit action potentials and transformed the discharge pattern from phasic discharge into a stimulus-dependent tonic discharge.

FIG. 3.

α-DTX alters membrane resonance and the characteristics of subthreshold oscillations. A: application of 100 nM α-DTX reduced the amplitude and frequency of subthreshold oscillations. B: membrane potential response to computer-generated impedance amplitude profile (ZAP) input current stimulus before and during toxin application. C: the impedance–frequency relationship before and during toxin application. Note the transformation from a band-pass (resonance) to a low-pass filter after toxin application. Holding potential −50 mV. D: in some neurons that did not exhibit bursting, application of 5 nM α-DTX resulted in rhythmical bursting in response to maintained depolarization. E: bursting was associated with an increase in the amplitude of the subthreshold oscillations after toxin application. F: impedance–frequency plot in response to ZAP input current stimulus obtained at −50-mV membrane potential.

FIG. 4.

Computer simulation replicates induction of bursting in nonbursting neuron. A: simulation of nonbursting neuron by increasing gKv1 by 100%(gKv1 = 12 arbitrary units) with maintained gNaP. Note spike discharge at the onset of the pulse only. Normal bursting is reinstated when the gKv1 conductance is lowered by 50% (gKv1 = 6), as expected. Inset: range of gKv1 conductances for bursting to occur. When the conductance is below a critical point, tonic discharge occurs, and when it is too high, complete block occurs. C and D are the same as A and B except a nonbursting neuron was simulated by lowering the gNaP by 6% and reinstating bursting by lowering gKv1 by 50% (gKv1 = 3). The bottom traces show the subthreshold oscillations during the various conditions. Lowering gKv1 enhances the amplitude of the oscillations, as shown experimentally.

FIG. 5.

Characteristics of the α-DTX–sensitive current. A: potassium currents recorded from whole cell patches in response to a family of voltage step commands (protocol, bottom trace) in control and 100 nM α-DTX. The α-DTX–sensitive current was obtained by subtraction of current in α-DTX from control (currents are leak subtracted). B: composite current–voltage relationships for total current and α-DTX–sensitive and insensitive current components. C: composite summary of normalized conductance plot for α-DTX–sensitive current (n = 6). The superimposed continuous curve is a Boltzmann fit to data points. D: plot of percentage of α-DTX–sensitive steady-state current component as a function of voltage. E: dose–response relationship for suppression of the low threshold Kv1 currents by α-DTX. F: example deactivation of α-DTX–sensitive currents. Command protocol is below current traces. G: deactivation time constant (tau) vs. membrane potential (n = 6).

FIG. 6.

Specific Kv1 peptide toxins block Kv1 current. A–D: plot of steady-state low threshold current vs. time after application of specific toxins. Each point represents steady-state current in response to a voltage step pulse from −90 to −40 mV before, during, and after application of either α-DTX, dendrotoxin-K (DTX-K), rTityustoxin-Kα (TiTx), or r-margatoxin (MgTX). Note the different time scales. Inset: toxin-sensitive current obtained by subtraction. E: summary bar chart (mean ± SE) of percent change of low threshold current amplitude in response to different subunit specific toxins. *Significant different between toxin and α-DTX (P < 0.05, 1-way ANOVA and Bonferonni post hoc analysis).

FIG. 7.

Effects of simultaneous application of specific subunit toxins on low-threshold current. A: example of time course of total low threshold current recorded during TiTX (100 nM) application and followed by α-DTX (100 nM). Note that the additional block by α-DTX is less than that by α-DTX alone (see Fig. 6E). B and C: summary plots of percentage reduction of total current by initial application of a specific toxin followed by subsequent application of another toxin in the same cell. Each point and connected line represents a single experiment. Black circles represent initial toxin application, whereas open circles indicate subsequent combined application.

FIG. 8.

Kv1 mRNA and protein expression in Mes V neurons. A: representative gel showing mRNA transcripts for Kv1.1 (441 bp) and Kv1.2 (458 bp) subunits in addition to the housekeeping gene GAPDH (452 bp) in the same neuron. B: bar chart indicating percentage of cells showing positive band for each subunit (numbers indicate cells observed). C: bar chart showing the percentage of neurons that expressed each number of subunits. D: immunohistochemical staining of Mes V neurons for Kv1.1, Kv1.2, and Kv1.6 showed translation of Kv1 mRNA subunits into functional proteins. ×40 objective, calibration bar indicates 100 μm.