Gating modes of calcium‐activated chloride channels TMEM16A and TMEM16B

Abstract

Key pointsr Calcium-activated chloride channels TMEM16A and TMEM16B support important physiological processes such as fast block of polyspermy, fluid secretion, control of blood pressure and sensory transduction.r Given the physiological importance of TMEM16 channels, it is important to study how incoming stimuli activate these channels. Here we study how channels open and close and how the process of gating is regulated.r We show that TMEM16A and TMEM16B display fast and slow gating. These gating modes are regulated by voltage and external chloride.r Dual gating explains the complex time course of the anion current. r Residues within the first intracellular loop of the channel influence the slow gating mode. r Dual gating is an intrinsic property observed in endogenous calcium-activated chloride channels and could be relevant to physiological processes that require sustained chloride ion movement.Abstract TMEM16A and TMEM16B are molecular components of the physiologically relevant calcium-activated chloride channels (CaCCs) present in many tissues. Their gating is dictated by membrane voltage (V m ), intracellular calcium concentrations ([Ca 2+ ] i ) and external permeant anions. As a consequence, the chloride current (I Cl ) kinetics is complex. For example, TMEM16A I Cl activates slowly with a non-mono-exponential time course while TMEM16B I Cl activates rapidly following a mono-exponential behaviour. To understand the underlying mechanism responsible for the complex activation kinetics, we recorded I Cl from HEK-293 cells transiently transfected with either TMEM16A or TMEM16B as well as from mouse parotid acinar cells. Two distinct V m -dependent gating modes were uncovered: a fast-mode on the millisecond time scale followed by a slow mode on the second time scale. Using long (20 s) depolarizing pulses both gating modes were activated, and a slowly rising I Cl was recorded in whole-cell and inside-out patches. The amplitude of I Cl at the end of the long pulse nearly doubled and was blocked by 100 μM tannic acid. The slow gating mode was strongly reduced by decreasing the [Cl − ] o from 140 to 30 mM and by altering the sequence of the first intracellular loop. Mutating 480 RSQ 482 to AVK in the first intracellular loop of TMEM16B nearly abolished slow gating, but, mutating 448 AVK 451 to RSQ in TMEM16A has little effect. Deleting 448 EAVK 451 residues in TMEM16A reduced slow gating. We conclude that TMEM16 CaCCs have intrinsic V m -and Cl − -sensitive dual gating that elicits complex I Cl kinetics.

Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models

“…Normalization was carried out using either G +120mV or G max to obtain G Norm (G/G max or G/ G +120mV ), which is proportional to the apparent open probability of the channel. We extracted the time constants by fitting the data to a mono or bi-exponential function of the form: $$y_0+a^{-t/\tau }\text{italic or }{y}_0+a^{-t/{\tau }_f}+b^{-t/{\tau }_s}$$where y 0 is the value of y when t→∞, t is the time, τ is a time constant, a and b are the contribution of fast and slow processes, and τ f and τ s are the fast and slow time constants [11], respectively. The dose-response curves were constructed using normalized currents; only patches with stable basal current obtained with 0 [Ca 2+ ] i were used.…”

“…This V m -dependence is explained by a voltage-dependent Ca 2+ dissociation [49]. We have shown that TMEM16A and TMEM16B exhibit fast and slow gating modes [11]. Both modes depend on intracellular Ca 2+ , membrane voltage (V m ) and the extracellular Cl − concentration ([Cl − ] o ).…”

Structural basis for phospholipid scrambling in the TMEM16 family

“…The basis for this effect is currently unknown since the location of the mutation is remote from the Ca 2+ binding site. Still, it is noteworthy that this residue is located in proximity to a stretch of amino acids that in TMEM16A was identified to influence Ca 2+ sensitivity [55,58,59]. The major question of how Ca 2+ ions activate scrambling is currently unknown.…”

Substituted 2-Acylaminocycloalkylthiophene-3-carboxylic Acid Arylamides as Inhibitors of the Calcium-Activated Chloride Channel Transmembrane Protein 16A (TMEM16A)

“…20 The kinetics of TMEM16A and TMEM16B Cl − conductance are complex, with distinct fast and slow gating mechanisms, regulated by membrane voltage and Cl − concentration. 21 Biophysical experiments suggest TMEM16A is present in membranes as a homodimer, 22, 23 with the dimerization domain located in the N-terminal sequence. 24 Recently, the C-terminal region of TMEM16A was modified to produce a constitutively active channel, providing a strategy to design TMEM16A activators.…”

“…26 Using the nhTMEM16 structure, two homology models of TMEM16A suggest it contains ten transmembrane helical segments, 21, 25 revising earlier eight helix structural models.…”