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. Nov-Dec 2009;2(11-12):612-9.
doi: 10.1242/dmm.003582. Epub 2009 Sep 24.

Episodic Ataxia Type 1 Mutations Differentially Affect Neuronal Excitability and Transmitter Release

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Free PMC article

Episodic Ataxia Type 1 Mutations Differentially Affect Neuronal Excitability and Transmitter Release

Joost H Heeroma et al. Dis Model Mech. .
Free PMC article

Abstract

Heterozygous mutations of KCNA1, the gene encoding potassium channel Kv1.1 subunits, cause episodic ataxia type 1 (EA1), which is characterized by paroxysmal cerebellar incoordination and interictal myokymia. Some mutations are also associated with epilepsy. Although Kv1.1-containing potassium channels play important roles in neuronal excitability and neurotransmitter release, it is not known how mutations associated with different clinical features affect the input-output relationships of individual neurons. We transduced rat hippocampal neurons, which were cultured on glial micro-islands, with lentiviruses expressing wild-type or mutant human KCNA1, and injected either depolarizing currents to evoke action potentials or depolarizing voltage commands to evoke autaptic currents. alpha-Dendrotoxin and tetraethylammonium allowed a pharmacological dissection of potassium currents underlying excitability and neurotransmission. Overexpression of wild-type Kv1.1 decreased both neuronal excitability and neurotransmitter release. By contrast, the C-terminus-truncated R417stop mutant, which is associated with severe drug-resistant EA1, had the opposite effect: increased excitability and release probability. Another mutant, T226R, which is associated with EA1 that is complicated by contractures and epilepsy, had no detectable effect on neuronal excitability; however, in common with R417stop, it markedly enhanced neurotransmitter release. The results provide direct evidence that EA1 mutations increase neurotransmitter release, and provide an insight into mechanisms underlying the phenotypic differences that are associated with different mutations.

Figures

Fig. 1.
Fig. 1.
Kv1.1 manipulation affects excitability. (A) Lentiviral expression of Kv1.1 does not alter overall Kv1.1 distribution in neurons after 15 days in vitro. CT, WT, RX and TR neurons stained for Kv1.1 (red, second column) and the dendrite marker microtubule associated protein 2 (MAP2) (magenta, third column). Transduction was verified by green fluorescent protein (GFP) expression (first column), which was driven by a separate promoter. Kv1.1 was primarily present in the perikaryal and somato-dendritic compartments but was also found in axons (defined by the absence of MAP2 staining, fifth column). Aggregation of endogenous Kv1.1 in the endoplasmic reticulum (ER) was not observed in any group. Bar, 25 μ m (somato-dendritic panels), 120 μ m (axonal panels). (B) WT and RX Kv1.1 have opposite effects on neuronal excitability. Example traces from CT neurons and neurons expressing WT, RX and TR Kv1.1 at 50%, 100% and 150% of the current threshold. The scale bar applies to all traces. (C) Cumulative probability plot showing an increase in the current threshold (normalized by capacitance) for neurons expressing WT Kv1.1 (black, n=43) compared with CT neurons (gray, n=29), and a reduction in the current threshold for RX-expressing neurons (red, n=36) compared with CT, WT and TR (blue, n=27) neurons. Inset: mean±s.e.m. current threshold/capacitance (using the same color code), based on the results of the Mann-Whitney U-test (*P≤0.05, **P≤0.01, ***P≤0.001). (D) TEA (1 mM) had no consistent effect on the current threshold in any group. (E) α-DTX (100 nM) reduced the current threshold (normalized by capacitance) in both WT- and RX-expressing neurons (paired t-tests).
Fig. 2.
Fig. 2.
Effects of Kv1.1 manipulation on action potentials. (A) Cumulative probability plot showing the distribution of firing rates measured at 150% of the current threshold (inset: mean±s.e.m.). WT-expressing neurons (n=16) showed an increased spike frequency compared with CT (n=15), RX (n=15) and TR (n=7) neurons. (B) WT-expressing neurons showed a decreased delay to the first spike (29±6 ms) compared with RX (78±7 ms, P<0.001), CT (63±11 ms, P=0.01) and TR (56±12 ms, P=0.04) neurons (Mann-Whitney U-tests; *P≤0.05, **P≤0.01, ***P≤0.001). (C) Sample traces from CT, WT, RX and TR neurons before, during and after TEA (1 mM) application. (D) Application of TEA increased the action potential duration in CT (n=10), WT (n=20), TR (n=8) and especially RX (n=16) neurons. (E) α-DTX (100 nM) did not affect spike width (Student’s t-tests; *P≤0.05, **P≤0.01, ***P≤0.001).
Fig. 3.
Fig. 3.
Effects of Kv1.1 manipulation on neurotransmitter release. (A) Upper trace: a 20-Hz spike train elicited in an autaptic neuron, showing four depolarizing stimulation artifacts (upward deflections) followed by escape action currents and EPSCs, respectively (downward deflections). Lower trace: a full 20-Hz train with stimulation artifacts and escape currents removed, showing 40 consecutive EPSCs. Inset: a schematic representation of an autaptic neuron. (B) Superimposed, successive autaptic responses from individual neurons obtained during a 40-pulse train at 5, 10 and 20 Hz (left, middle and right columns, respectively). At 5 Hz, the CT autapses (top row) showed relatively little synaptic rundown, but at 10 and 20 Hz, autaptic responses become gradually smaller owing to depletion of the RRP. WT neurons (second row) showed less rundown. RX (third row) and TR (fourth row) neurons showed increased rundown. (C) The RRP was estimated by back-extrapolating the steady-state replenishment phase to the ordinate. PRves was estimated as the ratio of the first response to the RRP. PPR was calculated as the ratio of the second to the first response.

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