Abstracts

Rationale: Nav1.1 and Nav1.2 are two of the main voltage-gated sodium channel subtypes expressed in central neurons. It is likely that there is a functional separation of these subtypes in that Nav1.1 is thought to be the predominant type in rapid spiking interneurons and Nav1.2 in pyramidal neurons. Sodium channels can enter the classical fast inactivated state as well as one or more slow inactivated states. The dynamics of entry into and exit from these states functionally affects action potential. Many anti-epileptic drugs such as phenytoin (PHT) are believed to modulate Nav channel inactivation. Characterisation of the inactivation properties and drug responsiveness of these Nav subtypes may clarify important therapeutic issues such drug refractoriness and paradoxical exacerbation of seizures with anti-epileptic medications. Methods: HEK293T cells expressing human Nav1.1 and Nav1.2 alpha subunits were used. Sodium currents were recorded in the whole cell configuration under voltage clamp. Conventional activation, inactivation, equilibrium, dynamic transitions between inactivated states as well as the effects of PHT on the properties of inactivation were examined. Results: n=68. Steady state inactivation (h ) parameters were measured with 50, 500 and 10000ms conditioning pulses (CP s). Interestingly, increasing duration of CP s resulted in a negative shift in the h curves, which were similar for both channel types (V0.5(50ms) -63.8 2.7, -64.6 1.6, V0.5(500ms) -70.8 2.6, -74.1 2.0, V0.5 (10000ms) -75.5 3.2, -78.9 3.0 for Nav1.1 and 1.2 respectively). The proportion of channels transferred to inactivated states with longer pulses was quite different between the channels subtypes (Nav1.1, n=11; Nav1.2, n=9). Nav1.1 had about 40% less channels (n=7) inactivated with transitions from -70 to -100 mV and more Nav1.2 channels entered inactivation with potentials changes from -100 to -60, -70, and -80 mV (n=7). Nav1.1 was less affected by use-dependent inactivation elicited by 40 Hz depolarization (n=6). Additionally, Nav1.1 (n=5) had a smaller proportion of channels were transferred to the slow inactivated state with 10s pulses compared with Nav1.2 (n=3). PHT had little effect on steady-state inactivation with 50 and 500ms CP s but cause a 5 to 6mV negative shift of V0.5 in 10000ms CP on both channel subtypes but more so with Nav1.1 (Nav1.1, n=3; Nav1.2, n=4). PHT slowed the time constants of use-dependent inactivation at both 25 and 40Hz on both channel subtypes without obvious differences between the two subtypes. Dose-response study had found that IC50 of Nav1.1 is higher than 1.2 which translated into a 26 M difference. Conclusions: Nav1.1 channels are less affected by slow inactivation processes and use dependent inactivation than Nav1.2. These differences may underlie for the high frequency firing behavior seen in the majority of interneurons which have a predominance of Nav1.1 subtype. PHT shifted the 10s CP h V0.5 by ~5mV in both channel types. Our data suggested PHT affecting slower inactivation processes in Nav1.2. The difference in IC50 of PHT is intriguing.