Biophysical and pharmacological properties of sodium and chloride channels reconstituted from embryonic rat brain growth cones

The biophysical and pharmacological properties of sodium (Na{dollar}sp+{dollar}) and chloride (Cl{dollar}sp-{dollar}) channels were examined following their reconstitution into planar lipid bilayers. A preparation of vesicular membrane fragments of embryonic rat brain growth cones (GCPs) was used as a source of Na{dollar}sp+{dollar} and Cl{dollar}sp-{dollar} channels for most experiments.; An examination of the guanidinium toxin pharmacology of single Na{dollar}sp+{dollar} channels derived from GCPs revealed two populations of channels with respect to mean K{dollar}sb{lcub}rm d{rcub}{dollar} values for saxitoxin (and decarbamoylsaxitoxin) block. The difference in K{dollar}sb{lcub}rm d{rcub}{dollar} values between the two populations of Na{dollar}sp+{dollar} channels was due entirely to a lower rate of toxin binding in the low affinity population. By comparison, mature rat brain synaptosomes contained only one population of channels with a K{dollar}sb{lcub}rm d{rcub}{dollar} for saxitoxin comparable to the high affinity GCP population.; When Na{dollar}sp+{dollar} channels were probed with C1-saxitoxin, a net neutral charged toxin, two populations of channels in the GCPs were no longer distinguishable. It was thus concluded that the differential presence of fixed anionic surface charges on the Na{dollar}sp+{dollar} channels from the GCPs accounts for the observed differences in the distribution of affinities for charged guanidinium toxins.; Chloride channels were also reconstituted from GCPs. It was noted, based on the relative frequency of appearance of channels in the lipid bilayers, that the density of Cl{dollar}sp-{dollar} channels in the GCPs was roughly 275 times that of mature rat brain synaptosomes. These Cl{dollar}sp-{dollar} channels were small conductance, outwardly rectifying channels of the "leak" or "background" type commonly found in secretory epithelial cells. Inhibition by the common stilbene and monocarboxylic acid inhibitors of Cl{dollar}sp-{dollar} channels was demonstrated. The gating and ion permeation characteristics of these channels were studied in detail.; Finally, the inhibition of reconstituted colonic epithelial Cl{dollar}sp-{dollar} channels by crude venom of the scorpion Leiurus quinquestriatus was demonstrated. The active component of the venom was purified to near homogeneity. The primary amino acid sequence of the 4070 Da peptide was determined, and found to have considerable homology to a class of previously described insect toxins. The purified ligand was found to block Cl{dollar}sp-{dollar} channels in a voltage-dependent manner with a K{dollar}sb{lcub}rm d{rcub}{dollar} of {dollar}approx{dollar}600 nM at 0 mV, and to induce a contracture paralysis when injected into arthropods. The possible mechanism of the observed toxicity to arthropods is discussed in the context of the known existence of small conductance Cl{dollar}sp-{dollar} channels in arthropod muscle.