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The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the
Valentina Cenedese1, Giulia Betto, Fulvio Celsi
1Neurobiology Sector, International School for Advanced Studies, and Italian Institute of Technology, SISSA Unit, 34136 Trieste, Italy.
Abstract:
Ca(2+)-activated Cl(-) channels (CaCCs) are involved in several physiological processes. Recently, TMEM16A/anoctamin1 and TMEM16B/anoctamin2 have been shown to function as CaCCs, but very little information is available on the structure-function relations of these channels. TMEM16B is expressed in the cilia of olfactory sensory neurons, in microvilli of vomeronasal sensory neurons, and in the synaptic terminals of retinal photoreceptors. Here, we have performed the first site-directed mutagenesis study on TMEM16B to understand the molecular mechanisms of voltage and Ca(2+) 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 Ca(2+) concentrations. We mutated E367 into glutamine or deleted the five consecutive glutamates (386)EEEEE(390) and (399)EYE(401). The EYE deletion did not significantly modify the apparent Ca(2+) dependence nor the voltage dependence of channel activation. E367Q and deletion of the five glutamates did not greatly affect the apparent Ca(2+) affinity but modified the voltage dependence, shifting the conductance-voltage relations toward more positive voltages. These findings indicate that glutamates E367 and (386)EEEEE(390) 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.
Insights
Mutations in the TMEM16B channel
Area of Science:
- Molecular biology
- Neuroscience
- Ion channel physiology
Background:
- Calcium-activated chloride channels (CaCCs) are crucial for physiological functions.
- TMEM16A and TMEM16B (anoctamins) are identified as CaCCs, but their structure-function relationships are poorly understood.
- TMEM16B is specifically localized in olfactory sensory neurons, vomeronasal sensory neurons, and retinal photoreceptors.
Purpose of the Study:
- To investigate the structure-function relationships of TMEM16B.
- To elucidate the molecular mechanisms underlying the voltage and calcium dependence of TMEM16B.
- To perform the first site-directed mutagenesis study on TMEM16B.
Main Methods:
- Site-directed mutagenesis was used to alter specific amino acids in the first intracellular loop of TMEM16B.
- Wild-type and mutant TMEM16B channels were expressed in HEK 293T cells.
- Whole-cell voltage-clamp electrophysiology was employed with varying intracellular calcium concentrations to analyze channel properties.
Main Results:
- Mutating E367 to glutamine (E367Q) or deleting the EEEEE motif (residues 386-390) significantly altered the voltage dependence of TMEM16B.
- These mutations shifted the conductance-voltage relationships towards more positive voltages.
- Deletion of the EYE motif (residues 399-401) did not substantially affect Ca(2+) or voltage dependence.
Conclusions:
- Glutamate residues E367 and the EEEEE motif in the first intracellular loop are critical for the voltage dependence of TMEM16B.
- This study provides initial insights into the structure-function of TMEM16B, particularly its voltage sensitivity.
- The findings contribute to understanding the molecular basis of CaCC function in sensory neurons and photoreceptors.
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