Burley, John Russell; (1997) Pharmacological and biophysical characteristics of voltage-dependent calcium channels in rat cerebellar granule neurones. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Voltage-dependent Ca2+ channels were investigated in rat cerebellar granule neurones using the whole-cell variant of the patch-clamp technique. Some biophysical properties were studied and a pharmacological approach was used to characterise the component parts of the whole-cell Ca2+ channel current using a selection of naturally occurring toxins, Ca2+ channel agonists, and antagonists. The ω-CTx MVIIC-, ω-Aga IVA- and ω-CgTx GVIA-sensitive components were found to account for 45% of the total whole-cell current and these toxins exhibited overlapping sensitivity to subtypes of the Ca2+ channel current in these cells. Low doses (1μM) of the DHP antagonists nicardipine inhibited a component of the current insensitive to the toxins used and accounted for 16% of the total current, whereas high doses (100 μM) caused inhibition of a greater proportion of the current than could be solely attributed to L-type Ca2+ channels. The current blocked by nicardipine in granule cells may be largely the result of L-type current, alternatively it may be blocking a non-L-type, DHP-sensitive channel. Experiments were conducted on a relatively recently cloned Ca2+ channel known to be expressed in granule cells. Transient expression of the α1E subunit was studied in the mammalian cell line COS-7. The sensitivity of α1E\β1b\α2δ channels to the DHP antagonist nicardipine and the agonist s-(-)-BayK8644 was determined. These channels were found to be inhibited by nicardipine but insensitive to s-(-)-BayK8644. The biophysical and pharmacological profile of this channel clone suggest that it has properties that do not fall into the existing classification of Ca2+ channel types. The α1E channel shares some features of both LVA and HVA Ca2+ channels. Changes in intracellular free Mg2+ caused a concentration-dependent reduction in whole-cell conductance. Some properties of currents were studied in the presence of different [Mg2+]i and changes in the pharmacological components of the current were apparent. A selective, but incomplete reduction in the ω-CgTx GVIA-sensitive current was shown. An estimation of the resting [Mg2+]i in cerebellar granule cells using the Mg2+-sensitive indicator MagFURA2 was 0.49 mM. A glutamate-induced increase in [Mg2+]i was studied in granule cells loaded with MagFURA2.

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