C. B. Gundersen

Glutamate and kainate receptors induced by rat brain messenger RNA in Xenopus oocytes

Xenopus laevis oocytes injected with poly(A)+ mRNA extracted from rat brain became sensitive to serotonin, glutamate, kainate, acetylcholine and γ-aminobutyrate. Application of these substances to mRNA-injected oocytes elicited membrane currents. The glutamate- and acetylcholine-induced currents usually showed oscillations, while the kainate current was smooth. The current oscillations during glutamate application reversed direction at about the chloride equilibrium potential (— 24 mV), but the reversal potential for the kainate current was close to 0 mV. The current-voltage relation for the glutamate-induced current oscillations showed strong rectification at hyperpolarized potentials, while that for the kainate current was nearly linear. In some oocytes, glutamate elicited smooth membrane currents, with oscillations either absent, or appearing after a delay. The reversal potential of this component was close to 0 mV, and was clearly different from that of the oscillatory component. The appearance of glutamate and kainate sensitivity in the oocyte membrane is due to the translation of the foreign messenger RNA, and not to activation of the oocytes’ own genome, because oocytes still become sensitive when transcription is prevented by enucleation or by treatment with actinomycin D. It appears that mRNA from rat brain contains translationally active messengers which code for various neurotransmitter receptors. When this mRNA is injected into Xenopus oocytes, the messengers are translated and receptors are inserted into the oocyte membrane, where they form functionally active receptor-channel complexes.

Serotonin receptors induced by exogenous messenger RNA in Xenopus oocytes

When poly(A)+-mRNA, extracted from rat brain, was injected into Xenopus laevis oocytes, it induced the appearance of serotonin receptors in the oocyte membrane. Application of serotonin to injected oocytes elicited, after a long delay, oscillations in membrane current. The equilibrium potential of this current corresponded with the chloride equilibrium potential. It appears that rat brain mRNA encodes the translation of serotonin receptors into the oocyte membrane. The combination of serotonin with these receptors leads to the opening of membrane channels.

Voltage-Operated Channels Induced by Foreign Messenger RNA in Xenopus Oocytes

Poly(A)+ messenger RNA (mRNA) extracted from rat brains or from cat muscles was injected into Xenopus laevis oocytes. This led to the incorporation of voltage-operated Na+ and K+ channels into the oocyte membrane. These channels are not normally present in the oocyte and presumably result from the synthesis and processing of proteins coded by the injected mRNA. Tetrodotoxin blocked the Na+ channels induced by mRNA derived from either innervated or denervated muscle.

The Antagonism between Botulinum Toxin and Calcium in Motor Nerve Terminals

The effects of tetraethylammonium and manganese, which modify calcium entry into motor nerve terminals, have been studied during advanced stages of botulinum paralysis. Evidence has been obtained that the voltage-activated calcium current in the nerve endings is not significantly reduced by botulinum toxin. The depression of transmitter release that the toxin produces must arise at a later stage, at an intracellular site of the release mechanism.

A transient inward current elicited by hyperpolarization during serotonin activation in Xenopus oocytes.

Activation of serotonin, glutamate or muscarinic receptors, incorporated into the membrane of Xenopus oocytes following injection of messenger RNA from rat brain, caused the development of a transient inward (Tin) current when the membrane was hyperpolarized. A detailed study was made of the Tin current induced during serotonin activation. The current is due principally to efflux of chloride ions, and is presumably activated by an influx of calcium ions, because it was blocked by removal of calcium from the bathing medium, by addition of manganese, cobalt or lanthanum, or by intracellular injection of EGTA. During application of serotonin, the amplitude of the Tin current increased slowly, and after washing it persisted for longer than the direct serotonin-induced current. The amplitude of the Tin current was sensitive to temperature and pH, and was abolished at pH 6.5 or by cooling to 12°C. The Tin current may be of importance in regulating the excitability of neurons in the central nervous system.

Properties of Human Brain Glycine Receptors Expressed in Xenopus oocytes

Glycine and γ-aminobutyric acid (GABA) receptors from the foetal human brain were ‘transplanted’ into the Xenopus oocyte membrane by injecting the oocytes with poly(A)+-mRNA extracted from the cerebral cortex. Activation of both glycine and GABA receptors induced membrane currents carried largely by chloride ions. However, unlike the GABAactivated current, the glycine current was blocked by strychnine, and was not potentiated by barbiturate. At low doses, the glycine current increased with concentration following a 2.7th power relation, suggesting that binding of three molecules of glycine may be required to open a single membrane channel. The current induced by steady application of glycine decreased with hyperpolarization beyond about —60 mV.

Intracellular Ca2+-dependent and Ca2+-independent responses of rat brain serotonin receptors transplanted to Xenopus oocytes

Xenopus oocytes injected with messenger RNA extracted from rat brain are induced to acquire a variety of neurotransmitter receptors and voltage-operated membrane channels. Activation of the receptors to serotonin, acetylcholine (muscarinic) and glutamate elicits oscillatory membrane currents carried by chloride ions. These currents are not abolished by removing external calcium, but are completely abolished after EGTA is injected into the oocytes to chelate intracellular calcium. A smooth current response to serotonin remained in EGTA-loaded oocytes, indicating that this response does not require intracellular calcium. In contrast to the oscillatory chloride currents, the chloride currents activated by GABA or glycine are not abolished by intracellular injection of EGTA. Thus, there appear to be two classes of chloride channels one of which requires intracellular calcium to open.

Slowly inactivating potassium channels induced in Xenopus oocytes by messenger ribonucleic acid from Torpedo brain.

Poly(A+) messenger RNA was extracted from the electric lobe and medulla of Torpedo and injected into oocytes of Xenopus laevis. The synthesis and processing of proteins coded by the injected messenger RNA led to the incorporation of voltage‐activated channels in the oocyte membrane. A large, well maintained outward current was recorded from injected oocytes in response to depolarization. Non‐injected oocytes did not show this current. The reversal potential of the current varied according to the Nernst equation with external potassium concentration, indicating that it was largely carried by potassium ions. The maintained potassium current was not reduced by manganese (5 mM) or lanthanum ions (0.1 mM). Tetraethylammonium and aminopyridines blocked the potassium current. The block produced by 3,4‐diaminopyridine was enhanced by previous activation, but diminished by strong depolarization. The amplitude of the potassium current increased over the approximate voltage range ‐30 to +50 mV, but reduced at more positive potentials. The decline of the current during maintained depolarization became slower as the membrane potential was made more positive, and the rate of onset of the current became faster. Estimates from noise analysis indicated that the slow potassium current passes through channels with a mean lifetime of about 14 ms and conductance of 14 pS (at ‐10 mV and room temperature). Injection of the messenger RNA also induced the formation of fast sodium and potassium channels activated by voltage, and channels activated by kainate.

The Reduction of Endplate Responses by Botulinum Toxin

Endplate responses were recorded in frog muscle fibres during an advanced stage of botulinum (BoTX) paralysis, when transmitter release had fallen to a very low level. By simultaneous recording from two points, it was found that, even when the quantal responses had been reduced to less than 0.01 per impulse (that is, four to five orders of magnitude below normal), the release continued to be spatially dispersed along the terminal arborization. These observations make it very unlikely that whole ‘active zones’ could be eliminated, as has been suggested, in all-or-none fashion by local action of BoTX molecules, and they suggest a more graded, indirect mechanism by which the toxin molecules interfere with the sites of transmitter release.

Expression of ACh-activated channels and sodium channels by messenger RNAs from innervated and denervated muscle

Xenopus oocytes were used to express polyadenylated messenger RNAs (mRNAs) encoding acetylcholine receptors and voltage-activated sodium channels from innervated and denervated skeletal muscles of cat and rat. Oocytes injected with mRNA from denervated muscle acquired high sensitivity to acetylcholine. whereas those injected with mRNA from innervated muscle showed virtually no response. Hence the amount of translationally active mRNA encoding acetylcholine receptors appears to be very low in normally innervated muscle, but increases greatly after denervation. Conversely. voltage-activated sodium currents induced by mRNA from innervated muscle were about three times larger than those from denervated muscle; this result suggests that innervated muscle contains more mRNA coding for sodium channels. The sodium current induced by mRNA from denervated muscle was relatively more resistant to block by tetrodotoxin. Thus a proportion of the sodium channels in denervated muscle may be encoded by mRNAs different from those encoding the normal channels.