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Putative pore‐loops of TMEM16/anoctamin channels affect channel density in cell membranes
Author(s) -
Adomaviciene Aiste,
Smith Keith J.,
Garnett Hannah,
Tammaro Paolo
Publication year - 2013
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2013.251660
Subject(s) - gating , transmembrane channels , biophysics , ion channel , phospholipid scramblase , chemistry , transmembrane protein , heterologous expression , microbiology and biotechnology , membrane protein , biology , membrane , biochemistry , voltage gated ion channel , receptor , recombinant dna , phospholipid , phosphatidylserine , gene
Key points• The recently identified TMEM16/anoctamin protein family includes Ca 2+ ‐activated Cl − channels (TMEM16A and TMEM16B), a Ca 2+ ‐activated non‐selective cation channel (TMEM16F) and proteins for which the function remains unclear. • TMEM16 channel proteins consist of eight putative transmembrane domains (TMs) with the 5th and 6th TMs flanking a loop predicted to protrude deep into the membrane. Recent studies suggest that this re‐entrant loop may compose part of the pore of TMEM16A channels while also containing residues involved in Ca 2+ binding. • Here, we investigate the functional role of the putative pore‐loop by examining the electrophysiological properties of chimeras produced by transplanting this region between TMEM16 family members with different conduction properties and Ca 2+ sensitivities. • We revealed that the putative pore‐loop of TMEM16 channels has an unexpected role in controlling the whole‐cell Ca 2+ ‐activated Cl − conductance by regulating the number of functional channels present on the plasma membrane.Abstract The recently identified TMEM16/anoctamin protein family includes Ca 2+ ‐activated anion channels (TMEM16A, TMEM16B), a cation channel (TMEM16F) and proteins with unclear function. TMEM16 channels consist of eight putative transmembrane domains (TMs) with TM5–TM6 flanking a re‐entrant loop thought to form the pore. In TMEM16A this region has also been suggested to contain residues involved in Ca 2+ binding. The role of the putative pore‐loop of TMEM16 channels was investigated using a chimeric approach. Heterologous expression of either TMEM16A or TMEM16B resulted in whole‐cell anion currents with very similar conduction properties but distinct kinetics and degrees of sensitivity to Ca 2+ . Furthermore, whole‐cell currents mediated by TMEM16A channels were ∼six times larger than TMEM16B‐mediated currents. Replacement of the putative pore‐loop of TMEM16A with that of TMEM16B (TMEM16A‐B channels) reduced the currents by ∼six‐fold, while the opposite modification (TMEM16B‐A channels) produced a ∼six‐fold increase in the currents. Unexpectedly, these changes were not secondary to variations in channel gating by Ca 2+ or voltage, nor were they due to changes in single‐channel conductance. Instead, they depended on the number of functional channels present on the plasma membrane. Generation of additional, smaller chimeras within the putative pore‐loop of TMEM16A and TMEM16B led to the identification of a region containing a non‐canonical trafficking motif. Chimeras composed of the putative pore‐loop of TMEM16F transplanted into the TMEM16A protein scaffold did not conduct anions or cations. These data suggest that the putative pore‐loop does not form a complete, transferable pore domain. Furthermore, our data reveal an unexpected role for the putative pore‐loop of TMEM16A and TMEM16B channels in the control of the whole‐cell Ca 2+ ‐activated Cl − conductance.