Calcium-activated chloride channels (CaCC) are broadly expressed in eukaryotes but their molecular identity remained enigmatic for over a score of years. In 2008 our group and two other groups reached the same conclusion that TMEM16A forms CaCC. Our study further showed that TMEM16B also forms CaCC. Molecular identification of CaCC as two members of the TMEM16 family of "transmembrane proteins with unknown function" has enabled us to approach questions concerning how CaCC works and how CaCC contributes to neuronal signaling in the brain. We propose to approach the following questions: How does calcium activate CaCC? Having found that TMEM16A-CaCC can be activated by a variety of divalent cations but is insensitive to dominant negative mutant calmodulin (CaM) expression or application of anti-CaM antibody that can reduce TRPV1 channel tachyphylaxis, we took on the task for a systematic mutagenesis survey of acidic residues for their possible involvement in calcium binding or gating. To this end, we first showed that a Drosophila TMEM16 family member forms CaCC. We then mutagenized several dozens of highly conserved acidic residues to identify acidic residues important for calcium gating. By varying the side chain at these positions and examining the mutant channel sensitivity to divalent cations of different size and chemistry, we will test the hypothesis that a subset of thes acidic residues coordinates calcium whereas others may provide second-shell carboxylates that interact with divalent cations. This study may also identify residues involved in the transduction of calcium gating. How does CaCC interact with permeant ions to facilitate chloride ion permeation? Having made the surprise finding that TMEM16F forms a small-conductance calcium-activated non-selective cation channel (SCAN), we looked for amino acids that are different between TMEM16A-CaCC and TMEM16F-SCAN, and found that mutagenesis of two residues in two separate transmembrane (TM) segments alter ion selectivity. Moreover, in collaboration with Dr. Min Li at Johns Hopkins, we have finished high throughput screening of >300,000 compounds to identify novel CaCC blockers. We will use novel CaCC pore blockers and thiol reagents in our mutagenesis studies to identify and characterize potential pore-lining residues. How might CaCC be involved in the calcium modulation of neuronal signaling? We found broad expression of TMEM16B but not TMEM16A in the brain. To generate TMEM16B knockout mice, we knocked in the coding sequence for farnesylated mCherry as a reporter for TMEM16B expression. Having found high expression of TMEM16B in the inferior olive implicated in cerebellar ataxia, dystonia, essential tremor and other disorders such as fetal alcohol syndrome, we will test for TMEM16B-CaCC involvement in the spike waveform and subthreshold oscillation and their modulation in inferior olive neurons.
Calcium-activated chloride channels (CaCCs) serve important physiological functions including modulation of neuronal signaling. Having established the TMEM16 family of transmembrane proteins of unknown function as a novel ion channel family that includes TMEM16A and TMEM16B as CaCC, we propose to use heterologous expression systems to study how CaCC channels work. We further generated TMEM16B knockout mice to examine the physiological role of TMEM16B in calcium modulation of neuronal signaling. We found that TMEM16B is highly expressed in the inferior olive neurons. Their action potential waveform and subthreshold oscillations are critically dependent on dendritic and somatic calcium channels and subjected to modulation by serotonin and NMDA receptors - processes that will elevate internal calcium levels. Given the involvement of the inferior olive in cerebella ataxia, dystonia, essential tremor of the elderly, and other disorders such as fetal alcohol syndrome and fatal insomnia, it will be important to understand how CaCC contributes to calcium modulation of inferior olive neuronal signaling.
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