Ca2+-activated c1- channels are involved in several physiological processes.In vertebrate olfactory transduction a Ca2+-dependent er efflux greatly amplifies the odorant response. The binding of odorants to receptors in the cilia of olfactory sensory neurons activates a transduction cascade that involves the opening of cyclic nucleotide-gated channels and the entry of ca2+ in the cilia. ca2+ activates a c1· current that, in the presence of a maintained elevated intracellular c1· concentration, produces an efflux of er ions and amplifies the depolarization. TMEM16A/anoctamin1 and TMEM16B/anoctamin2 have been shown to function as CaCCs. TMEM16B is expressed in the cilia of olfactory sensory neurons, microvilli of vomeronasal sensory neurons, and in the synaptic terminals of retinal photoreceptors, but very little information is available on the structure-function relation. Here we have performed the first site-directed mutagenesis study on TMEM16B to understand the molecular mechanisms of voltage- and Ca2+- dependence. We have mutated amino acids in the first putative intracellular loop and measured the properties of the wild type and mutant TMEM16B channels expressed in HEK 293T cells using the whole-cell voltage-clamp technique in the presence of various intracellular ca2+ concentrations. Mutation of E367 into glutamine or deletion of five consecutive glutamates 3s6EEEEE390 did not greatly affect the apparent ca2+ affinity, but modified the voltage dependence shifting the conductance-voltage relations toward more positive voltages. These findings indicate that glutamates E367 and 3s6EEEEE390 in the first intracellular putative loop play an important role in the voltage-dependence of TMEM16B, thus providing an initial structure-function study for this channel.
|Titolo:||The voltage-dependence of TMEM16B/anoctamin2: the calcium-activated chloride channel in olfactory transduction|
|Data di pubblicazione:||3-apr-2012|
|Appare nelle tipologie:||8.1 PhD thesis|