Die Rolle von TRPC1 in CA1-Neuronen des murinen Hippocampus

The classical or canonical "transient receptor potential" channels (TRPC1 - TRPC7) are a family of nonselective cation channels, that are activated in a phospholipase C-dependent manner and are expressed in many tissues, e. g. the brain (cortex, hippocampus, etc.; e. g. Chung et al., 2006). TRPC channels are thought to be tetrameric, and the assembly of different TRPC subunits into homo- and heteromultimers creates channels with different regulatory and physiological properties. In heterologous expression systems, TRPC1 heteromultimerizes with and modifies the properties of TRPC4 and TRPC5 (Strübing et al., 2001). In CA1 hippocampal neurons TRPC1, TRPC4 and TRPC5 are coexpressed and in these neurons, the activation of Gq-coupled receptors induces a Ca2+-activated nonselective cation current (ICAN; Congar et al., 1997). TRPC channels have been suggested to be involved in the generation of this current, but there is little hard evidence to support this. Therefore, the aim of this thesis was to characterize the properties of the cation current activated by stimulation of metabotropic glutamate receptors of type I (mGluRI) by (RS)-3,5-dihydroxyphenylglycine (DHPG) in CA1 neurons of the murine hippocampus and to study the role of TRPC1 in the generation of this current by comparing the responses from wild type (TRPC1+/+) and homozygous TRPC1 knockout mice (TRPC1-/-). TRPC1-/-mice show no obvious phenotype (Dietrich et al., 2007), and the morphology of the hippocampus is largely unchanged. At the microscopical level, Golgi-stained CA1 neurons showed a small but significant increase in the number of primary branches. In whole-cell voltage-clamp recordings, the stimulation of mGluRI led to the development of a current in WT as well as in TRPC1-/- mice. The current-voltage relationship of the DHPG-sensitive current was S-shaped with a minimum at around -50 mV, a maximum at +40 mV and a reversal potential near -10 mV. The inward current was carried by extracellular cations like Na+ and Ca2+ and was modulated by the intracellular Ca2+ as well as the extracellular Mg2+ concentration. Interestingly, the ICAN in CA1 neurons was voltage-dependent. Therefore, the nonselective cation channel required activation of mGluRI and membrane depolarization to be activated, acting as a coincidence detector. The major difference between TRPC1+/+ and TRPC1-/- neurons was a significant increase of the inward current in TRPC1-/- mice. Another finding was that the increase of the inward current in TRPC1-/- mice was detectable two weeks after birth. The larger current in TRPC1-/- mice is unlikely to result from a change in the expression patterns of TRPC isoforms. Expression levels of TRPC mRNAs in the whole hippocampus measured using quantitative real-time PCR were, with the exception of TRPC1, similar in WT and TRPC1-/- mice. To investigate the physiological consequences of the increased DHPG-sensitive inward current in TRPC1-/- mice, whole-cell current-clamp recordings were performed. CA1 neurons from WT and TRPC1-/- mice showed a comparable resting membrane potential. In most experiments, the stimulation of mGluRI in WT induced membrane depolarization leading to the generation of, or an increase in spike activity. In contrast, in TRPC1-/- mice, stimulation by DHPG led to stronger membrane depolarizations and to an increased tendency of these neurons to show so-called epileptiform activity associated with the generation of plateau potentials. In response to small current injections in the presence of the mGluRI agonist, TRPC1+/+ neurons showed a weak slow afterdepolarization or a stronger depolarization to a plateau potential which persisted up to 55 s following the current injection. In addition to these patterns, neurons in TRPC1-/- mice mainly showed plateau potentials similar to that in TRPC1+/+ mice, but with a significantly shorter duration. In conclusion, this study showed that TRPC1 has a negative regulatory role in CA1 hippocampal neurons where it reduces the inward nonselective cation current generated in response to mGluRI stimulation. This probably occurs as an effect of the formation of heteromultimeric channels with TRPC4 and TRPC5. Deletion of TRPC1 results in an increase of the number of neurons developing a plateau potential in response to simultaneous membrane depolarization and mGluRI activation. Since plateau potentials may be involved in the generation of epileptiform activity, the physiological role of TRPC1 may be the modulation of cell excitability in CA1 hippocampal neurons.