Millar, Julie Anne; (2000) Characterisation of a novel potassium conductance in rat cerebellar granule neurons. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cultured cerebellar granule neurons (CGNs) possess transient and delayed rectifier type voltage-gated potassium (K+) conductances. An additional component of outward current has been described in these cells which has been termed standing outward current (IKso). This current is outwardly rectifying, non-inactivating and is reversibly and concentration dependently inhibited by muscarine. The aim of this study was to characterise a number of biophysical and pharmacological properties of IKso in an attempt to identify the molecular correlate of the current and elucidate the mechanism of muscarinic modulation. The effect of M2 and M3 muscarinic receptor antagonists on the muscarine concentration response curve was determined. The M2 antagonist (methoctramine) had little effect on the control concentration response curve, while the same concentration of a M3 antagonist (zamifenacin) produced a rightward shift identifying the M3 receptor subtype as mediating the muscarine effect. (Additional concentrations of zamifenacin resulted in an estimated pA2 value of 8.13). Inhibiting a classical downstream product of M3 receptor activation (PLC), only slightly reduced the muscarinic inhibition of IKso, suggesting that M3 receptors may act through a novel pathway to inhibit IKso in CGNs. It has been proposed that the molecular correlate of IKso may be a member of the ether à go go (eag) family of K+ channels, since vat-eag (r-eag) channels when expressed in mammalian cells show similar properties to IKso. A feature of v-eag currents is a dramatic slowing of the activation kinetics on application of external Mg2+ in a concentration and voltage dependent manner. The activation kinetics of IKso were found to be unaffected by external Mg2+, arguing against eag being the molecular correlate of IKso. IKso also shares certain functional properties with members of the two pore domain superfamily of K+ channels (KT). A lack of voltage dependence of activation has been demonstrated for IKso, which is a diagnostic feature of KT channels. Pharmacologically IKso is inhibited by Ba2+, NMDG, external acidification, is weakly inhibited by quinine and quinidine, but is unaffected by arachidonic acid. These properties mean IKso bears closest resemblance to the KT clone TASK-1. The properties of IKso are almost identical to those of TASK-1, and RT-PCR revealed mRNA for TASK-1 is expressed in CGNs. Additionally immunocytochemical experiments confirmed the presence of TASK-1 protein in both the membrane and cytoplasm of the cells. It seems likely therefore that IKso belongs to the KT superfamily of potassium channels.

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