Veratridine modification of the purified sodium channel alpha-polypeptide from eel electroplax.

Abstract

In the interest of continuing structure-function studies, highly purified sodium channel preparations from the eel electroplax were incorporated into planar lipid bilayers in the presence of veratridine. This lipoglycoprotein originates from muscle-derived tissue and consists of a single polypeptide. In this study it is shown to have properties analogous to sodium channels from another muscle tissue (Garber, S. S., and C. Miller. 1987. J0urna/ of General Physiology. 89:459-480), which have an additional protein subunit. However, significant qualitative and quantitative differences were noted. Comparison of veratridine-modified with batrachotoxin-modified eel sodium channels revealed common properties. Tetrodotoxin blocked the channels in a voltage-dependent manner indistinguishable from that found for batrachotoxin-modified channels. Veratridine-modified channels exhibited a range of single-channel conductance and subconductance states. The selectivity of the veratridine-modified sodium channels for sodium vs. potassium ranged from 6-8 in reversal potential measurements, while conductance ratios ranged from 12-15. This is similar to BTX-modified eel channels, though the latter show a predominant single-channel conductance twice as large. In contrast to batrachotoxin-modified channels, the fractional open times of these channels had a shallow voltage dependence which, however, was similar to that of the slow interaction between veratridine and sodium channels in voltage-clamped biological membranes. Implications for sodium channel structure are discussed.

Effects of veratridine on sodium currents and fluxes

“…Na ÷ channels incorporated into lipid bilayers with the aid of veratridine show clear voltage-dependent block by TTX with Kd increasing with depolarization. This has been reported for electroplax channels (Duch et al 1989) as well as those from lobster walking leg nerves. In both preparations batrachotoxin-modified channels are also blocked in a potential-dependent fashion (Levinson et al 1986;Castillo et al 1992).…”

“…Thus in the rare case of one-channel patches the potential at which fo=0.5 varied by 30 mV. In other cases the results are complicated since depolarization also increases the number of conducting channels (Duch et al 1989). Membranes with single lobster nerve channels show closures on the order of milliseconds (fast process), seconds (slow process), and minutes, leading to fast and slow types of fo that increased with depolarization and have different midpoint potentials of -122 and -95 mV, respectively.…”

“…Veratridinemodified channels in natural membranes show lower conductances (see previous section), most probably because they have been determined at lower (natural) cation concentrations. In some preparations conductance sublevels are found in the presence of veratridine, for example, in eel electroplax (Duch et al 1989); this may, however, result from unresolved rapid channel closings. Also, some rat brain channels modified by veratridine or batrachotoxin have another lower conductance state (ca.…”

Alkaloid-modified sodium channels from lobster walking leg nerves in planar lipid bilayers.

“…Vr values were interpolated using the data from +30 to -30 mV and given with respect to the chamber containing only NaCI (extracellular aspect of the channel). electroplax (KI/2 = 17.5 nM; a = 0.53; Duch et al, 1989) and the TIX K1/2 for sodium channels from lobster leg nerves determined with flux studies (12 nM; Barnola and Villegas, 1976).…”

Isolation of two saxitoxin-sensitive sodium channel subtypes from rat brain with distinct biochemical and functional properties

“…Gordon et al (1987) demonstrated that this rat brain Na channel preparation contained 18% R I Na channel and some mixture of Rn and Rin Na channels by immunological criteria (the antibodies used to distinguish R n Na channels would be expected to crossreact with RHI since the antigen had 75% homology between these two types). Although there have been other reports in the literature of low conductance BTX-and VER-activated channels from brain Green et al, 1987;Duch et al, 1988Duch et al, , 1989, it has not been clear whether these low conductance states are due to multiple substates, of a single channel or multiple channel types.…”