FGF14 modulates resurgent sodium current in mouse cerebellar Purkinje neurons
- Duke University Medical Center, United States
Figures
Figure 1
FGF14 is a component of the NaV1.6 macromolecular complex in mouse cerebellum, but shRNA knockdown does not affect NaV current density in cultured cerebellar Purkinje neurons.
(A) Co-immunoprecipitation (IP) of NaV1.6 by FGF14 from mouse cerebellum and immunoblot (IB) with the indicated antibodies. Mw markers are indicated on right. Arrowheads indicate NaV1.6 (top panel) …
Figure 2
FGF14 knockdown in cerebellar Purkinje neurons affects multiple NaV channel biophysical properties.
(A and B) Voltage-dependence of NaV channel activation and steady-state inactivation in cerebellar Purkinje neurons transfected with Scrambled control shRNA (Scrambled), FGF14 shRNA (shRNA), or …
Figure 3
Expression of the dominant negative FGF14ΔNT affects multiple NaV channel biophysical properties indicating essential roles for the FGF14 N-terminus.
(A) Schematic of endogenous wild type (WT) FGF14 binding to the C-terminus of the NaV1.6 α subunit (top) and magnified schematic of the expressed FGF14ΔNT preventing binding of the WT FGF14 (in …
Figure 4
The FGF14bR/A mutant that prevents interaction with the NaV C-terminus cannot rescue NaV kinetic effects of FGF14 knockdown.
(A) Surface representation of the crystal structure of a ternary complex containing the NaV1.5 C-terminus domain (CTD, green), FGF13 (yellow), and calmodulin (purple); the interaction surfaces are …
Figure 5
Action potential dynamics and repetitive spiking are dependent upon FGF14 and its N-terminus.
(A) Overlay of single action potentials evoked with a 10 ms current injection (current amplitude shown in parentheses). (B) Current threshold to induce action potentials for the indicated …
Tables
Table 1
NaV current activation and inactivation parameters in cerebellar Purkinje neurons
| Activation | Inactivation | |||||
|---|---|---|---|---|---|---|
| V1/2 (mV) | K | n | V1/2 (mV) | K | n | |
| Scrambled | −34.4 ± 1.5 | 4.0 ± 0.3 | 21 | −49.7 ± 1.2 | 4.2 ± 0.1 | 19 |
| shRNA | −35.0 ± 1.9 | 4.3 ± 0.5 | 22 | −59.0 ± 1.2** | 4.7 ± 0.2 | 20 |
| shRNA/FGF14bWT | −35.5 ± 1.5 | 3.9 ± 0.3 | 14 | −52.0 ± 0.9 | 5.2 ± 0.3 | 9 |
| shRNA/FGF14RA | −33.2 ± 1.5 | 3.5 ± 0.2 | 12 | −50.9 ± 1.4 | 4.4 ± 0.3 | 11 |
| FGF14bWT | −33.1 ± 1.4 | 4.5 ± 0.5 | 14 | −50.6 ± 1.3 | 4.9 ± 0.3 | 10 |
| FGF14∆NT | −33.9 ± 2.0 | 3.9 ± 0.5 | 14 | −59.1 ± 0.8** | 4.7 ± 0.3 | 15 |
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Mean ± s.e.m. (n), **p < 0.01 compared to Scrambled control.
Table 2
Intrinsic membrane properties and single action potential (AP) characteristics measured in cerebellar Purkinje neurons
| Scrambled | shRNA | shRNA/FGF14bWT | shRNA/FGF14bRA | FGF14∆NT | |
|---|---|---|---|---|---|
| Input resistance (MΩ) | 221.8 ± 15.8 (7) | 238.0 ± 49.5 (11) | 266.6 ± 24.5 (11) | 225.9 ± 10.8 (6) | 269.5 ± 30.2 (6) |
| Resting membrane potential (mV) | −54.5 ± 1.3 (21) | −55.8 ± 1.2 (19) | −54.4 ± 1.0 (16) | −55.4 ± 0.7 (10) | −54.9 ± 1.4 (11) |
| Current threshold (pA) | 77.8 ± 6.4 (21) | 142.1 ± 18.5 (19)** | 78.8 ± 10.4 (16) | 170.5 ± 16.7 (10)** | 191.0 ± 22.1 (11)** |
| AP threshold (mV) | −35.6 ± 1.0 (21) | −32.8 ± 1.2 (19) | −34.5 ± 0.8 (16) | −32.8 ± 1.4 (10) | −32.7 ± 0.8 (11) |
| AP amplitude (mV) | 78.8 ± 3.2 (21) | 77.5 ± 2.8 (19) | 78.9 ± 4.5 (16) | 79.1 ± 4.3 (10) | 73.1 ± 4.7 (11) |
| AP duration (ms) | 2.3 ± 0.2 (21) | 2.0 ± 0.1 (19) | 2.2 ± 0.1 (16) | 1.8 ± 0.1 (10) | 2.1 ± 0.3 (11) |
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Mean ± s.e.m. (n), **p < 0.01 compared to Scrambled control.
