A pore for TMEM16a

TMEM16a is a Ca2+-activated Cl- channel that whose molecular identity was recently uncovered in 2008 by three independent labs, including the lab of Lily Jan at UCSF. Thanks to the recent crystal structure of the fungal homolog nhTMEM16, we now know where calcium binds to TMEM16a to regulate channel activity; however, the pore region remains elusive.

In the recent manuscript Peters et al,  Lily Jan and colleagues tackle identification of the TMEM16a pore.  To do so, the authors identified twelve basic, positively charged, amino acids that could potentially line the pore of TMEM16a. They created various channel mutants by mutating these basic residues to alanines. Since the channel passes negatively charged chloride ions, they believed that disrupting the interaction between the positive regions of basic residues would impact the flow of ions within the channel.Changing the charge of pore lining residues is expected to alter channel permeation. With this method, the authors identified five residues; R511A, K599A, R617A, K641A, and R784A that produced a significant shift in the permeability ratios of Br– compared to Cl–.  Three of these residue, R511A, K599A, and R617A, also produced a shift in the permeability ratio of SCN– (thiocynate) compared to Cl–, thus implying that these mutations increase selectivity for larger anions; such a finding would be expected when substituting residues with bulky side chains such as arginine and lysine, with residues alanine, which has a much smaller side chain.

The authors also found that TMEM16a permeation changes by varying intracellular calcium concentration. In particular, channels with the R511A, K599A, R617A, and R784A mutations, they passed large anions, such as SCN–, at high intracellular calcium concentrations where they would normally show higher Cl– selectively. Together, these data suggest that these four basic residues play and important role in the permeation of ions through TMEM16a and could contribute to the pore region. These residues cluster around a subunit cavity on the extracellular domain TMEM16a.

Figure from Peters et al., 2015: Homology model of TMEM16a with the basic residues thought to line the pore of TMEM16a

In more directly test the role that these four residues may play in the TMEM16a’s pore, the authors identified two channel blockers that were effective at preventing the permeation of ions through wild type channels. However, these channel blockers did not produce the same effect when exposed to mutant channels. The effectiveness of the blockers was increased in two mutant channels and almost eliminated in the other two mutant channels. The results further demonstrate the importance of these basic residues in the pore region of TMEM16a.

Overall, this paper identifies some of the important putative-pore lining residues involved in ion permeation of TMEM16a. By doing so, this paper provides some of the first pieces of information about the ion conducting pore of TMEM16a despite the lack of structural information for such an important ion channel.

Post by Sarah Sokol and Anne Carlson