Effects of FGF14 and NaVß4 deletion on transient and resurgent Na current in cerebellar Purkinje neurons.

Voltage-gated Na channels of Purkinje cells are specialized to maintain high availability during high-frequency repetitive firing. They enter fast-inactivated states relatively slowly and undergo a voltage-dependent open-channel block by an intracellular protein (or proteins) that prevents stable fast inactivation and generates resurgent Na current. These properties depend on the pore-forming α subunits, as well as modulatory subunits within the Na channel complex. The identity of the factors responsible for open-channel block remains a question. Here we investigate the effects of genetic mutation of two Na channel auxiliary subunits highly expressed in Purkinje cells, NaVß4 and FGF14, on modulating Na channel blocked as well as inactivated states. We find that although both NaVß4 and the FGF14 splice variant FGF14-1a contain sequences that can generate resurgent-like currents when applied to Na channels in peptide form, deletion of either protein, or both proteins simultaneously, does not eliminate resurgent current in acutely dissociated Purkinje cell bodies. Loss of FGF14 expression does, however, reduce resurgent current amplitude and leads to an acceleration and stabilization of inactivation that is not reversed by application of the site-3 toxin, anemone toxin II (ATX). Tetrodotoxin (TTX) sensitivity is higher for resurgent than transient components of Na current, and loss of FGF14 preferentially affects a highly TTX-sensitive subset of Purkinje α subunits. The data suggest that NaV1.6 channels, which are known to generate the majority of Purkinje cell resurgent current, bind TTX with high affinity and are modulated by FGF14 to facilitate open-channel block.

New Cav2 calcium channel gating modifiers with agonist activity and therapeutic potential to treat neuromuscular disease.

Voltage-gated calcium channels (VGCCs) are critical regulators of many cellular functions, including the activity-dependent release of chemical neurotransmitter from nerve terminals. At nerve terminals, the Cav2 family of VGCCs are closely positioned with neurotransmitter-containing synaptic vesicles. The relationship between calcium ions and transmitter release is such that even subtle changes in calcium flux through VGCCs have a strong influence on the magnitude of transmitter released. Therefore, modulators of the calcium influx at nerve terminals have the potential to strongly affect transmitter release at synapses. We have previously developed novel Cav2-selective VGCC gating modifiers (notably GV-58) that slow the deactivation of VGCC current, increasing total calcium ion flux. Here, we describe ten new gating modifiers based on the GV-58 structure that extend our understanding of the structure-activity relationship for this class of molecules and extend the range of modulation of channel activities. In particular, we show that one of these new compounds (MF-06) was more efficacious than GV-58, another (KK-75) acts more quickly on VGCCs than GV-58, and a third (KK-20) has a mix of increased speed and efficacy. A subset of these new VGCC agonist gating modifiers can increase transmitter release during action potentials at neuromuscular synapses, and as such, show potential as therapeutics for diseases with a presynaptic deficit that results in neuromuscular weakness. Further, several of these new compounds can be useful tool compounds for the study of VGCC gating and function.