32 Winking, Mathis Kühn, Frank J. P. Spehr, Marc 512000-4 163310 160000 Publikationsserver der RWTH Aachen University Aachen German 114 S. : Ill., graph. Darst. TRP channels are polymodal receptors that are involved in many substantial physiological processes. TRPM8 is a sensor for cold temperatures, but can additionally be activated by substances that mediate a cold feeling such as menthol. Furthermore, the TRPM8 channel is voltage-dependent, whereby the voltage sensitivity can be positively modulated both by, cold as well as by various TRPM8 channel agonist. In contrast to the well studied "classical" voltage-dependent cation channels, there is no generally accepted model for TRPM8, which describes the gating mechanism in detail so far. Based on a recently published computer simulated model for TRPM8 gating, we used site directed mutagenesis to systematically analyze potential interactions between transmembrane domain S3 and S4, which has been demonstrated to be essential for voltage sensitivity. Furthermore, the structural and functional importance of S3 and S4 for gating and folding of TRPM8 was analyzed. In the present work, the evidence of a functional cooperation between the transmembrane segment S3 and S4 in TRPM8 was proofed. The charge distribution in the central region of these protein domains plays a crucial role for a proper folding and for posttranslational maturation (glycosylation) of the channel and therefor is essential for its functionality. Furthermore, the highly conserved sequence motif N-x-x-D located in S3, is not only for TRPM8 but also for the closely related TRPM2 channel of functional relevance. In addition, new insights could be gained about the voltage dependence of TRPM8 by systematic changes of charged amino acids within the S4 segment, which question the so far favored model of an autonomous mobile S4 voltage sensor in TRPM8. Taken together, the results of this study indicate a protein domain corporately formed by S3 and S4 in TRPM8. This domain conduces i.a. the interaction of the channel with different TRPM8 agonists and additionally is of crucial importance for the voltage sensitivity of the channel, possibly through an interaction with the negatively charged phospholipid PIP2. Aachen, Techn. Hochsch., Diss., 2014 ; PUB:(DE-HGF)11, ; 2, ; Spannungskontrollierter Ionenkanal Dissertation / PhD Thesis Diss. Aachen, Techn. Hochsch. 2015 2014 RWTH-CONV-207020 2015 https://publications.rwth-aachen.de/record/463095