Acidosis Differentially Modulates Inactivation in NaV1.2, NaV1.4, and NaV1.5 Channels

NaV channels play a crucial role in neuronal and muscle excitability. Using whole-cell recordings we studied effects of low extracellular pH on the biophysical properties of NaV1.2, NaV1.4, and NaV1.5, expressed in cultured mammalian cells. Low pH produced different effects on different channel subtypes. Whereas NaV1.4 exhibited very low sensitivity to acidosis, primarily limited to partial block of macroscopic currents, the effects of low pH on gating in NaV1.2 and NaV1.5 were profound. In NaV1.2 low pH reduced apparent valence of steady-state fast inactivation, shifted the τ(V) to depolarizing potentials and decreased channels availability during onset to slow and use-dependent inactivation (UDI). In contrast, low pH delayed open-state inactivation in NaV1.5, right-shifted the voltage-dependence of window current, and increased channel availability during onset to slow and UDI. These results suggest that protons affect channel availability in an isoform-specific manner. A computer model incorporating these results demonstrates their effects on membrane excitability

Chimeric Agents Derived from the Functionalized Amino Acid, Lacosamide, and the α-Aminoamide, Safinamide: Evaluation of Their Inhibitory Actions on Voltage-Gated Sodium Channels, and Antiseizure and Antinociception Activities and Comparison with Lacosamide and Safinamide

The functionalized amino acid, lacosamide ((R)-2), and the α-aminoamide, safinamide ((S)-3), are neurological agents that have been extensively investigated and have displayed potent anticonvulsant activities in seizure models. Both compounds have been reported to modulate voltage-gated sodium channel activity. We have prepared a series of chimeric compounds, (R)-7–(R)-10, by merging key structural units in these two clinical agents, and then compared their activities with (R)-2 and (S)-3. Compounds were assessed for their ability to alter sodium channel kinetics for inactivation, frequency (use)-dependence, and steady-state activation and fast inactivation. We report that chimeric compounds (R)-7–(R)-10 in catecholamine A-differentiated (CAD) cells and embryonic rat cortical neurons robustly enhanced sodium channel inactivation at concentrations far lower than those required for (R)-2 and (S)-3, and that (R)-9 and (R)-10, unlike (R)-2 and (S)-3, produce sodium channel frequency (use)-dependence at low micromolar concentrations. We further show that (R)-7–(R)-10 displayed excellent anticonvulsant activities and pain-attenuating properties in the animal formalin model. Of these compounds, only (R)-7 reversed mechanical hypersensitivity in the tibial-nerve injury model for neuropathic pain in rats

A single residue differentiates between human cardiac and skeletal muscle Na+ channel slow inactivation.

Slow inactivation determines the availability of voltage-gated sodium channels during prolonged depolarization. Slow inactivation in hNa(V)1.4 channels occurs with a higher probability than hNa(V)1.5 sodium channels; however, the precise molecular mechanism for this difference remains unclear. Using the macropatch technique we show that the DII S5-S6 p-region uniquely confers the probability of slow inactivation from parental hNa(V)1.5 and hNa(V)1.4 channels into chimerical constructs expressed in Xenopus oocytes. Site-directed mutagenesis was used to test whether a specific region within DII S5-S6 controls the probability of slow inactivation. We found that substituting V754 in hNa(V)1.4 with isoleucine from the corresponding position (891) in hNa(V)1.5 produced steady-state slow inactivation statistically indistinguishable from that in wild-type hNa(V)1.5 channels, whereas other mutations have little or no effect on slow inactivation. This result indicates that residues V754 in hNa(V)1.4 and I891in hNa(V)1.5 are unique in determining the probability of slow inactivation characteristic of these isoforms. Exchanging S5-S6 linkers between hNa(V)1.4 and hNa(V)1.5 channels had no consistent effect on the voltage-dependent slow time inactivation constants [tau(V)]. This suggests that the molecular structures regulating rates of entry into and exit from the slow inactivated state are different from those controlling the steady-state probability and reside outside the p-regions

Structural determinants of slow inactivation in human cardiac and skeletal muscle sodium channels.

Skeletal and heart muscle excitability is based upon the pool of available sodium channels as determined by both fast and slow inactivation. Slow inactivation in hH1 sodium channels significantly differs from slow inactivation in hSkM1. The beta(1)-subunit modulates fast inactivation in human skeletal sodium channels (hSkM1) but has little effect on fast inactivation in human cardiac sodium channels (hH1). The role of the beta(1)-subunit in sodium channel slow inactivation is still unknown. We used the macropatch technique on Xenopus oocytes to study hSkM1 and hH1 slow inactivation with and without beta(1)-subunit coexpression. Our results indicate that the beta(1)-subunit is partly responsible for differences in steady-state slow inactivation between hSkM1 and hH1 channels. We also studied a sodium channel chimera, in which P-loops from each domain in hSkM1 sodium channels were replaced with corresponding regions from hH1. Our results show that these chimeras exhibit hH1-like properties of steady-state slow inactivation. These data suggest that P-loops are structural determinants of sodium channel slow inactivation, and that the beta(1)-subunit modulates slow inactivation in hSkM1 but not hH1. Changes in slow inactivation time constants in sodium channels coexpressed with the beta(1)-subunit indicate possible interactions among the beta(1)-subunit, P-loops, and the slow inactivation gate in sodium channels