Electrophysiological characterization of the heat-gated ion channels: TRPV1, TRPV3 and TRPV4

The mammalian nervous system constantly probes both internal and external temperatures in order to avoid thermal extremes and regulate body temperature. Several members of the transient receptor potential (TRP) family of ion channels are temperature sensitive and expressed in tissues relevant to thermosensation. In the studies presented in Chapter 1, we demonstrated that TRPV4, previously demonstrated to be hypoosmotically gated, is a heat-gated channel and its activity can be regulated by changes in osmolarity. We highlighted that TRPV4 is gated by lower temperatures (>28°C) than a homologous heat-gated channel, TRPV1, which is activated by noxious temperatures (>42°C). We observed TRPV4-like immunoreactivity in skin keratinocytes and a region of the brain where thermal and osmotic information is integrated for proper homeostatic responses. Recent studies have demonstrated that TRPV4 is required for normal innocuous thermosensation in mice. These findings taken together suggest that in physiological settings TRPV4 is a warm-gated channel important role for thermosensation and possibly thermoregulation.; In order to identify regions within TRPV subfamily members that render these channels responsive to distinct temperatures, we hypothesized that the determinants of TRPV1-like heat response (>42°C activation threshold) is transferable to other homologous heat sensitive channels. In our mutagenic studies described in Chapter 2, we demonstrated that the large substitutions of TRPV1 cytosolic amino and carboxyl terminal domains onto TRPV4 are sufficient for mimicking TRPV1-like heat responses. Further studies will be required to pinpoint the roles of specific amino acids within these regions.; As described in Chapter 3, we have also demonstrated that TRPV l, TRPV2 and TRPV3 are activated by diphenylboronic anhydride (DPBA) and inhibited by diphenyltetrahydrofuran (DPTHF). However, when we applied a high concentration of DPBA to mammalian cells expressing TRPV3, we observed a multiphasic current response. Elongated heat applications also evoked a multiphasic current. Further electrophysiological studies revealed the existence of two states of activation that reflected a modification in the pore structure and an associated change in the ability of calcium to permeate the pore. We believe that this structural transformation leads to an apparent activity dependent loss of calcium inhibition and might be critical for the TRPV gating mechanism.