Action potential generation is controlled by the opening, inactivation, and recovery of voltage-gated sodium channels (Na V ). A-type fibroblast growth factor homologous factors (A-type FHFs), which are cytosolic Na V binding proteins, mediate rapid-onset, long-term inactivation (LTI) of Na V by contributing an independent inactivation particle that competes with the Na V intrinsic fast inactivation mechanism (Dover et al., J. Physiol. 588:3695; 2010) . To further explore the structural mechanism of A-type FHF induced LTI of Na V , we have performed cotransfection of wild-type and mutant FHF2A expression plasmids together with wild-type and mutagenized TTX-resistant Nav1.6 and Nav1.5 expression plasmids into Neuro2A cells, and have analyzed TTX-resistant sodium currents through various whole cell voltage clamp protocols. Our results demonstrate that LTI induced by FHF2A only occurs when the channels reach the open state. FHF2A can compete for binding to channels with another open channel blocking protein, Na V b 4, which is responsible for resurgent sodium current due to its rapid dissociation from channel upon repolarization. Both FHF2A and Na V b 4 blocking particles rely upon hydrophobic and basic residues, suggesting they may share a common binding surface within Na V 's cytoplasmic cavern exposed in the open state. However, FHF2A dissociates much more slowly from channels than does Na V b 4, accounting for their functional dissimilarity. Ongoing channel mutagenesis seeks to reveal channel binding sites for each of these blocking particles. In summary, our studies show that A-type FHFs serve as open sodium channel blockers, in competition with both the Na V intrinsic fast inactivation mechanism and the accessory subunit Na V b 4, to drive channels into a long-term refractory state. Experimental Approach A-Type Fibroblast Growth Factor Homologous Factors Function as Open Channel Blockers to Modulate Voltage-gated Sodium Channel Availability Yue Liu 1,2 , Mitchell Goldfarb 1 1 Department of Biological Sciences, Hunter College of City University, 695 Park Avenue, New York, NY 10065, USA 2 The Graduate Center Biology Program, City University of New York, New York, NY 10016, USA Voltage-gated sodium channels (Na V ) consist of a large a subunit, the core of the channel which can function on its own. The a subunit has four domains (I~IV), each containing six membrane-spanning regions (S1~S6). Na V Mediates inward current of action potentials upon membrane depolarization of excitable cells. Na V inactivation can be caused by its intrinsic fast inactivation particle and accessory proteins (A-type FHFs and Na V b 4). Here we study long-term inactivation of Na V a induced by FHF2A (one form of A-type FHFs) and the functional competition between these two accessory protein. Abstract Results Acknowledgement Supported by NIH/NIGMS R01-GM095430 Grateful to all members in Prof. Mitchell Goldfarb’s lab Introduction N-terminal hydrophobic and basic FHF2A residues mediate sodium channel long-term inactivation (A) N-terminal sequence of FHF2A and engineered FHF2A mutations . (B) Nenro2A cells transfected with Na V 1.6 +/- FHF2A and its mutants were subjected to 4 pulses of depolarization separated by 40 msec -90 mV intervals. Examples of mutants that reduce or eliminate long-term inactivation (LTI) accumulation. (C) Mutations of hydrophobic (upper panel) and basic (lower panel) amino acids in the N- terminal FHF2A reduce or eliminate LTI accumulation. All FHF2A mutants that reduce long-term inactivation accelerate sodium channel recovery (FHF2A dissociation) rate All FHF2A mutants that reduce long-term inactivation accelerate sodium channel recovery (FHF2A dissociation) rate All FHF2A mutants that reduce long-term inactivation accelerate sodium channel recovery (FHF2A dissociation) rate (A) Long-term recovery of Na V 1.6 in the cells co-expressing FHF2A (WT) (B) FHF2A (R11Q/R17Q) dissociation from Na V 1.6. FHF2A long-term inactivation particle is an open-channel blocker (A) Neuro2A cells transfected with Na V 1.6 (F1478Q) + FHF2A were depolarized to different test voltages to assay channel activation, each depolarization was followed by 40 msec -90 mV recovery and a second depolarization to 0 mV; current induced by second depolarization reported LTI induced by test voltage . LTI induction required activation of some channels, indicating open-channel block. (B) Voltage dependence of channel activation and LTI are very similar Summary FHF2A long-term inactivation and Na V b 4 blocking particles engage in reciprocal functional competition (A) Neuro2A cells expressing fast inactivation defective Na V 1.5 (F1486Q) were depolarized with 4 pulses of 48 msec +70 mV separated by 40 msec -100 mV intervals (E Na ≈-10 mV). F2A (2-18) peptide drives the channels into maximal LTI after the first pulse (upper-right). Na v β4 (154-167) peptide blocks the channels at +70 mV but dissociates rapidly upon repolarization to -100 mV, giving 4 cycles of resurgent currents (lower-left). With both F2A and b4 peptides, the channels undergo accumulated LTI, with gradually decreasing resurgent current (lower-right) . (B) LTI of Na V 1.5 (F1486Q) induced by F2A +/- β4 (C) Resurgent current by β4+/- F2A 1.A-type FHF long-term inactivation (LTI) particle functions as open-channel blocker. 2.A-type FHFs competes with another open- channel blocker Na v β4 functionally to modulate Na V availability. 3.N-terminal hydrophobic and basic residues of A- type FHFs contribute to Na V LTI. (C) Recovery of Na V 1.6 in the cells expressing different FHF2A mutants.