Tatulian, Lucine; (2002) Sodium and potassium ion channels as targets for the control of epilepsy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Epilepsy is one of the most common neurological disorders and affects roughly 1% of the population. Since the electrical hyperexcitability that causes epilepsy is directly created by currents flowing through ion channels, drugs that selectively block sodium channels, particularly persistent sodium currents, and drugs that open KCNQ potassium channels are a reasonable choice for treating epilepsy. In the first part of the present study, the whole-cell voltage clamp technique was used to study the biophysical and pharmacological properties of a fully inactivating sodium channel present in rat sympathetic neurones (SCG) and human Nav1.3 brain sodium channel expressed in Chinese hamster ovary (CHO) cells, capable of showing a persistent component. The principal aim of these experiments was to find out whether these currents could be modulated by certain transmitters and intracellular messengers. Molecular biology and immunocytochemistry were used to show that a combination of Nav1.1, Nav1.2, Nav1.3 and Nav .7 sodium channels contribute to the fully inactivating sodium current, recorded in SCG neurones. The biophysics of activation were similar for currents recorded in SCG neurones and CHO cells. Phorbol 12-myristate 13 acetate, an activator of PKC, decreased the peak amplitude of the sodium current in SCG neurones, but did not induce a persistent component. To see if a more physiological activation of PKC would reproduce this effect. M₁ muscarinic acetylcholine receptors and α₂ adrenoceptors were stimulated by oxotremorine-M and norepinephrine, respectively. This, however, had no effect on the sodium current. In CHO cells direct PKC stimulation by phorbol dibutyrate did not change the peak amplitude of either the transient or persistent components of Nav 1.3 current. Staurosporine, a non-selective inhibitor of protein kinases, did not change the transient or the persistent current amplitude in CHO cells. Finally, the effects of intracellular application of GTPγS were studied in SCG neurones and CHO cells. This did not have any effect on any of the currents. These results indicate that persistent sodium channels in these cells do not seem to be modulated by G-protein or PKC mediated pathways. The second part of the thesis concerns the action of retigabine. This is a novel anticonvulsant compound now in clinical phase II development. During the course of my studies, retigabine was shown to enhance currents generated by KCNQ2/3 potassium channels, which are considered to be molecular correlates of some mammalian neuronal M channels. In this study, I used the perforated patch clamp technique to compare the actions of retigabine on KCNQ2/3 currents, with those on currents generated by other members of the KCNQ family (homomeric KCNQ 1, 2, 3 and 4 channels) expressed in CHO hm1 cells, and on the native M current in rat sympathetic neurones. Retigabine (10 μM) increased current amplitudes in cells expressing KCNQ2, 3, 4 and 2/3 channels and shifted the voltage-dependence of channel activation. Maximum shifts (mV) were: KCNQ2: -24.2; KCNQ3: -42.8; KCNQ4: -24.6; KCNQ2/3: -30.4. EC₅₀ values for half-maximal shifts were (μM; mean ± s.e.m; (n)): KCNQ2: 2.5 ± 0.6 (5); KCNQ3: 0.6 ± 0.3 (3); KCNQ4: 5.2 ± 0.9 (3); KCNQ2/3: 1.9 ± 0.2 (5). In contrast, retigabine did not enhance cardiac KCNQ1 currents but instead, at high concentrations (100 μM), reduced currents recorded at strongly positive potentials. Retigabine also enhanced native M-type currents recorded from dissociated rat SCG neurones. At 10 μM, retigabine produced a 21 mV left-shift in the activation curve for the linopirdine-sensitive component of outward current. In unclamped neurones, retigabine produced a hyperpolarisation and reduced the number of action potentials produced by depolarising current injections, without change in action potential configuration. If replicated in central neurones, this could account for retigabine's anti-epileptic action.

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