Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Svobodova, B.
Exploring the molecular basis of the gating machinery of TRPC3 ion channel complex
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Graz Medical University; 2019. pp. 84
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Birner-Grünberger Ruth
- Groschner Klaus
- Schreibmayer Wolfgang
- Altmetrics:
- Abstract:
- TRPC proteins form unique class of cationic channels able to sense, integrate and relay multiple cellular signals. Despite constituting predominantly non-selective channels, their physiological relevance has been clearly linked with cellular Ca2+-dependent processes and their impact on membrane potentials in excitable cells. Recognition of their involvement in an increasing number of pathological states and diseases such as cardiac hypertrophy, arrhythmias, epilepsy, cerebellar ataxia, Huntington’s and Alzheimer’s disease, etc. calls for means of therapeutic intervention. Effective pharmacological control however requires detailed molecular information on the channel’s gating mechanism. The present study aims to identify and further describe the molecular elements of TRPC3 channel gating by physiological lipid stimuli and a recently discovered synthetic direct TRPC3/6 channel agonist, GSK1702934A (abb. GSK). Using site directed mutagenesis we identified a conserved glycine at position 652 in the S6 of TRPC3 that dramatically influences both activation modes of the channel. Our electrophysiological analysis showed strong reduction in response in TRPC3G652A channels to physiological stimulation via PLC pathway. TRPC3G652A also exhibited altered potency profile of exogenously administered lipids and various GSK structural analogues (benzimidazoles) in comparison to WT TRPC3 channels. Interestingly, the stimulation with benzimidazole compounds resulted in enhanced currents via TRPC3G652A channels. We proved that neither increased potency of benzimidazoles to TRPC3G652A channels or a modified pore architecture are responsible for that observation. Inspection of the gating properties at the single channel level further revealed a unique, long-lived open state in benzimidazole stimulated TRPC3G652A channels. Moreover, TRPC3G652A channels lacked PLC/diacylglycerol mediated cross-desensitization to GSK activation as usually observed for WT TRPC3 channels. Lack of desensitization in TRPC3G652A channels allowed for large benzimidazole-induced Ca2+ signals in conditions that fully desensitized WT TRPC3 channels. Collectively our data suggest, supported by the recent high-resolution cryo-EM based TRPC3 structure, that lipid and possibly also benzimidazole agonists share the recognition site localized to a lateral window between 2 adjacent TRPC3 subunits as well as certain common features of the TRPC3 gating machinery.