Fast inactivation of Nav1.3 channels by FGF14 proteins: An unconventional way to regulate the slow firing of adrenal chromaffin cells

Using Nav1.3 and FGF14 KO mice, Martinez-Espinosa et al. provide new findings on how intracellular FGF14 proteins interfere with the endogenous fast inactivation gating and regulate the “long-term inactivation” of Nav1.3 channels that sets Nav channel availability and spike adaptation during sustained stimulation in adrenal chromaffin cells.

A unique mechanism of inactivation gating of the Kv channel family member Kv7.1 and its modulation by PIP2 and calmodulin

Inactivation of voltage-gated K+ (Kv) channels mostly occurs by fast N-type or/and slow C-type mechanisms. Here, we characterized a unique mechanism of inactivation gating comprising two inactivation states in a member of the Kv channel superfamily, Kv7.1. Removal of external Ca2+ in wild-type Kv7.1 channels produced a large, voltage-dependent inactivation, which differed from N- or C-type mechanisms. Glu295 and Asp317 located, respectively, in the turret and pore entrance are involved in Ca2+ coordination, allowing Asp317 to form H-bonding with the pore helix Trp304, which stabilizes the selectivity filter and prevents inactivation. Phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+-calmodulin prevented Kv7.1 inactivation triggered by Ca2+-free external solutions, where Ser182 at the S2-S3 linker relays the calmodulin signal from its inner boundary to the external pore to allow proper channel conduction. Thus, we revealed a unique mechanism of inactivation gating in Kv7.1, exquisitely controlled by external Ca2+ and allosterically coupled by internal PIP2 and Ca2+-calmodulin.

Nav1.7 gating in human iPSC derived sensory neurons: an experimental and computational study

Chronic pain is a global healthcare problem with a huge societal impact. Its management remains unsatisfactory, with no single treatment clinically approved in most cases. In this study we use an in vitro experimental model of erythromelalgia consisting of sensory neurons derived from human induced pluripotent stem cells obtained from a patient (carrying the mutation F1449V) and a control subject. We combine neurophysiology and computational modelling to focus on the Nav1.7 voltage gated sodium channel, which acts as an amplifier of the receptor potential in nociceptive neurons and plays a critical role in erythromelalgia due to gain of function mutations causing the channel to open with smaller depolarisations.Using multi-electrode array (extracellular) recordings, we found that the scorpion toxin OD1 increases the excitability of sensory neurons in cultures obtained from the control donor, evidenced by increased spontaneous spike rate and amplitude. In erythromelalgia cultures, the application of the Nav1.7 blocker PF-05089771 effectively stopped spontaneous firing. These results, which are in accordance with current clamp and voltage clamp recordings reported in the literature, are explained with a conductance-based computational model of a single human nociceptive neuron. The disease was simulated through a decrease of the Nav1.7 half activation voltage, which decreased the rheobase and increased the response to supra threshold depolarizing currents. This enhanced response could be successfully supressed by blocking the Nav1.7 channels. The painful effects of OD1 were simulated through a slower establishment and a quicker removal of Nav1.7 inactivation, reproducing the effects of the toxin on the spike frequency and amplitude. Our model simulations suggest that the increase in extracellular spike amplitude observed in the MEA after OD1 treatment can be due mainly to a slope increase in the ascending phase of the intracellular spike caused by impaired inactivation gating.

Flow and shortcuts along the Shaker Kv channel slow inactivation gating cycle

Closing the cycle of Kv channel slow inactivation gating.

Rapid constriction of the selectivity filter underlies C-type inactivation in the KcsA potassium channel

C-type inactivation is a time-dependent process observed in many K+ channels whereby prolonged activation by an external stimulus leads to a reduction in ionic conduction. While C-type inactivation is thought to be a result of a constriction of the selectivity filter, the local dynamics of the process remain elusive. Here, we use molecular dynamics (MD) simulations of the KcsA channel to elucidate the nature of kinetically delayed activation/inactivation gating coupling. Microsecond-scale MD simulations based on the truncated form of the KcsA channel (C-terminal domain deleted) provide a first glimpse of the onset of C-type inactivation. We observe over multiple trajectories that the selectivity filter consistently undergoes a spontaneous and rapid (within 1–2 µs) transition to a constricted conformation when the intracellular activation gate is fully open, but remains in the conductive conformation when the activation gate is closed or partially open. Multidimensional umbrella sampling potential of mean force calculations and nonequilibrium voltage-driven simulations further confirm these observations. Electrophysiological measurements show that the truncated form of the KcsA channel inactivates faster and greater than full-length KcsA, which is consistent with truncated KcsA opening to a greater degree because of the absence of the C-terminal domain restraint. Together, these results imply that the observed kinetics underlying activation/inactivation gating reflect a rapid conductive-to-constricted transition of the selectivity filter that is allosterically controlled by the slow opening of the intracellular gate.

Inverted allosteric coupling between activation and inactivation gates in K+ channels

The selectivity filter and the activation gate in potassium channels are functionally and structurally coupled. An allosteric coupling underlies C-type inactivation coupled to activation gating in this ion-channel family (i.e., opening of the activation gate triggers the collapse of the channel’s selectivity filter). We have identified the second Threonine residue within the TTVGYGD signature sequence of K+ channels as a crucial residue for this allosteric communication. A Threonine to Alanine substitution at this position was studied in three representative members of the K+-channel family. Interestingly, all of the mutant channels exhibited lack of C-type inactivation gating and an inversion of their allosteric coupling (i.e., closing of the activation gate collapses the channel’s selectivity filter). A state-dependent crystallographic study of KcsA-T75A proves that, on activation, the selectivity filter transitions from a nonconductive and deep C-type inactivated conformation to a conductive one. Finally, we provide a crystallographic demonstration that closed-state inactivation can be achieved by the structural collapse of the channel’s selectivity filter.