Electrophysiological study of veratridine-modified rat brain type IIA sodium channels and their interactions with open channel blocking compounds

Abstract

Alkaloid toxins affecting electrical excitability have been known for about a century, with articles by Sydney Ringer describing their effects appearing in the first volume of the Journal of Physiology. Yet, the molecular mechanisms leading to these profound effects are almost completely unknown. An important aim in sodium channel electrophysiology and pharmacology is to understand these toxin-channel interactions at the molecular level.

The present thesis attempts to better describe the effects of one such alkaloid toxin, veratridine (VTD), on the -subunit of the rat brain type IIA (RIIA) sodium channel. Alkaloid toxins are known to affect interactions between channel-blocking compounds and channels. Experiments using open-channel blockers were designed to better understand this phenomenon.

The macroscopic voltage-dependent kinetics of VTD-modified RIIA Na channel -subunit expressed heterologously in CHO cells were studied. The activation and deactivation kinetics are well described by a double-exponential function but poorly by a mono-exponential function. The fast time constant and associated amplitude factor showed a steep potential dependence, unlike the slow component. The steady-state activation of VTD-modified channels is described by a Boltzmann function with V½ = -131.9 mV and a slope of 9.41 mV. A two-state model is proposed for the fast component that provides an explanation for the kinetic mechanism of action of veratridine.

With a detailed understanding of VTD effects on whole-cell RIIA Na currents, an effort was made to localize the VTD-binding site using open-channel blockers: KIFMK (a synthetic peptide), QX-314 (a quaternary analog of lidocaine), and tetrapentylammonium ions (TPeA).

The synthetic pentapeptide KIFMK causes a use- and voltage-dependent block of the RIIA sodium channel. Studies on the RIIA sodium channel expressed in CHO cells reveal that the fraction of VTD-modified channels decreases linearly with increasing KIFMK concentration. However, the time constant for dissociation from the channel remains unchanged in the presence of high concentrations of KIFMK, unlike in the presence of QX-314, where dissociation appears more complex. These data are consistent with mutually exclusive binding of the open-channel blocking peptide and veratridine to the brain sodium channel.

Experiments were undertaken to study the interaction of VTD-modified channels with TPeA, known to block open Na channels, to further understand the effects of VTD on open-channel block. TPeA blocks the brain sodium channel in a manner similar to the cardiac sodium channel. Removal of inactivation using chloramine-T (CT) unmasks a time-dependent block by TPeA, consistent with slow blocking kinetics. TPeA blocks CT-treated channels in a potential- and use-dependent manner, suggesting a role for the activation gate in use-dependence.

TPeA neither blocks nor significantly affects gating of VTD-modified channels. This indicates that VTD modifies the channel such that the TPeA-binding site is lost.

Conclusions from the results obtained are presented at the end of this chapter.