TY - JOUR
T1 - Ion channel and lipid scramblase activity associated with expression of TMEM16F/ANO6 isoforms
AU - Scudieri, Paolo
AU - Caci, Emanuela
AU - Venturini, Arianna
AU - Sondo, Elvira
AU - Pianigiani, Giulia
AU - Marchetti, Carla
AU - Ravazzolo, Roberto
AU - Pagani, Franco
AU - Galietta, Luis J V
PY - 2015/9/1
Y1 - 2015/9/1
N2 - TMEM16F, also known as ANO6, is a membrane protein that has been associated with phospholipid scramblase and ion channel activity. However, the characteristics of TMEM16F-dependent channels, particularly the ion selectivity, are a matter of debate. Furthermore, the direct involvement of TMEM16F in phospholipid scrambling has been questioned. We studied the properties of different TMEM16F variants generated by alternative splicing. Using whole-cell patch-clamp recordings, we found that V1, V2 and V5 variants generated membrane currents activated by very high (micromolar) intracellular Ca2+ concentrations and positive membrane potentials. These variants showed different degrees of Ca2+ sensitivity and kinetics of activation but similar ion permeability, characterized by a slight selectivity for Cl- over Na+. A fourth variant (V3) showing a unique carboxy-terminus was devoid of activity, in agreement with its intracellular localization. We also measured scramblase activity using the binding of annexin V to detect phosphatidylserine on the cell surface. V1, V2 and V5 variants were associated with calcium-dependent phosphatidylserine externalization. Interestingly, introduction of an activating mutation, D409G, produced a marked increase in the apparent Ca2+ sensitivity of TMEM16F-dependent channels. In parallel, this mutation also enhanced the extent of phosphatidylserine externalization that occurred even under resting conditions. These results support the conclusion that TMEM16F proteins are directly involved in dual activity, as a phospholipid scramblase and as an ion channel. Journal compilation
AB - TMEM16F, also known as ANO6, is a membrane protein that has been associated with phospholipid scramblase and ion channel activity. However, the characteristics of TMEM16F-dependent channels, particularly the ion selectivity, are a matter of debate. Furthermore, the direct involvement of TMEM16F in phospholipid scrambling has been questioned. We studied the properties of different TMEM16F variants generated by alternative splicing. Using whole-cell patch-clamp recordings, we found that V1, V2 and V5 variants generated membrane currents activated by very high (micromolar) intracellular Ca2+ concentrations and positive membrane potentials. These variants showed different degrees of Ca2+ sensitivity and kinetics of activation but similar ion permeability, characterized by a slight selectivity for Cl- over Na+. A fourth variant (V3) showing a unique carboxy-terminus was devoid of activity, in agreement with its intracellular localization. We also measured scramblase activity using the binding of annexin V to detect phosphatidylserine on the cell surface. V1, V2 and V5 variants were associated with calcium-dependent phosphatidylserine externalization. Interestingly, introduction of an activating mutation, D409G, produced a marked increase in the apparent Ca2+ sensitivity of TMEM16F-dependent channels. In parallel, this mutation also enhanced the extent of phosphatidylserine externalization that occurred even under resting conditions. These results support the conclusion that TMEM16F proteins are directly involved in dual activity, as a phospholipid scramblase and as an ion channel. Journal compilation
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U2 - 10.1113/JP270691
DO - 10.1113/JP270691
M3 - Article
AN - SCOPUS:84940612857
VL - 593
SP - 3829
EP - 3848
JO - Journal of Physiology
JF - Journal of Physiology
SN - 0022-3751
IS - 17
ER -