As ion channels go, TMEM16A are busy ones. As one of a number of channels responsible for chloride conduction at the cell surface, their activity has implications for both water movement and transmembrane potential. They are found in the cells of epithelial and smooth muscle tissue throughout the body, and their functional diversity encompasses secretion, cell proliferation, cardiac excitability, smooth muscle contraction and the prevention of polyspermy. With such a broad range of locations and potential functions, it stands to reason that their control mechanism might be complex. Indeed, if you look them up in the ion channel and receptor guide published by the BJP, they are not readily categorised as either ‘ligand-gated’ or ‘voltage-gated’, but languish under the heading ‘other’ alongside several other recent additions to the chloride channel family (ClC, CFTR and volume-regulated channels). Since their molecular identification in 2008, investigation into their gating control has generated a complex and sometimes confused picture involving both ligand and voltage mechanisms. A recent paper by Contreras-Vite and colleagues2 attempts to integrate experimental evidence gained over the last 8 years in the proposal of an updated model of TMEM16A gating.
Factors at play in TMEM16A activation
There are several well-established factors controlling the conduction of chloride ions through TMEM16A channels. Primarily:
- TMEM16A is a chloride channel, activated directly by intracellular calcium
- Activation by calcium is strongly influenced by membrane potential
- Speed of opening/closing is influenced by the concentration and nature of the permeant anion
These first two factors are inextricably linked. Under ‘resting’ physiological conditions of intracellular Ca2+ concentration (0.1 uM) and membrane potential (-40 to -60 mV, for example), these channels appear to be closed despite the presence of calcium. Depolarisations above the chloride equilibrium potential begin to elicit a TMEM16A current, conduction increasing with increasing depolarisation, giving TMEM16A its classic ‘outward-rectifier’ profile. However, when intracellular calcium concentration increases beyond 1 uM, voltage sensitivity appears to be lost, and TMEM16A conduction is seen at negative and positive membrane potentials alike. There is also evidence to suggest that the intracellular side of the channel has the capacity to bind 2 Ca2+ ions. In terms of gating speed, both fast and slow gating kinetics have been seen (whole-cell and patch recordings) depending on the duration of membrane depolarisation. This speed also appears to be influenced by the level of extracellular chloride, with the slow component most markedly affected (slowed further) by increasing extracellular chloride levels from 30 to 140 mM. More permeant anions (SCN–, I–, NO3–) promote/accelerate opening and slow channel closure when applied extracellularly.
So how do you bring these factors together in order to model TMEM16A gating? In the present study2, Contreras-Vite and his colleagues look at their own experimental findings combined with published information, presenting for example the novel observation that in zero intracellular calcium, TMEM16A conduction is still possible, but requires strong depolarisations beyond +100 mV. They also show that reducing extracellular chloride reduces channel open probability, and any ‘fast’ gating kinetics are entirely lost when the channel is maximally activated by high levels of intracellular calcium, and state that intracellular chloride level appears to have no effect on channel activation.
They use these findings to calculate the open-probability of the channel under the influence of these different factors, and define the rate constants governing the transitions between discreet ‘open’ (O) and ‘closed’ (C) states when 0, 1 or 2 Ca2+ ions are bound to the channel in the presence or absence of 1 external Cl– ion. By using these to simulate steady-state activation properties and comparing these to their experimentally-derived activation and closure (tail-current) data, they came up with the following 12-state Markov chain model:
Essentially, ‘open’ channel states are represented in the right half of the model, ‘closed’ in the left, concentric levels represent calcium binding – from the outer level in which both putative Ca2+ binding sites are occupied, the centre representing the channel with no calcium bound; each state being linked by a rate constants representing parameters listed fully in the paper, most of which are voltage-dependent, some being fast and some being slow (indicated in the diagram key).
Using this model, the authors demonstrate that they can reproduce the activation and deactivation kinetics shown by their experimental data, although they themselves admit that the quality of the fit begins to decrease under extreme levels of intracellular calcium and voltage. They do, however, successfully use it to predict that calcium binding affinity does not change with varying extracellular chloride. They then show experimentally that this does appear to be the case.
The basis of this latest gating model comes from evidence which is only briefly summarised here. There are, of course, other factors which have been proposed to influence TMEM16A channel activity under physiological conditions, such as the binding of calmodulin and inhibition of activation by intracellular protons. Whether this model proves to be correct, time will tell. But in targeting drugs to this channel, knowing how stable and long-lasting some of these conformations may be under various physiological conditions might lead to more efficient, state-dependent drug pharmacology.
Blog written by Sarah Lilley
- “Still round the corner there may wait, A new road or a secret gate.” J R R Tolkein
- Contreras-Vite JA, Cruz-Rangel S, De Jesús-Pérez JJ, Figueroa IA, Rodríguez-Menchaca AA, Pérez-Cornejo P, Hartzell HC, Arreola J. (2016) Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models. Pflugers Arch. 2016 May 2. [Epub ahead of print]
- Cruz Rangel S, De Jesús Pérez JJ, Contreras Vite JA, Pérez Cornejo P, Hartzell H, Arreola J (2015) Gating modes of calcium-activated chloride channels TMEM16A and TMEM16B. JPhysiol 24:5283–98. doi:10.1113/JP271256, PMID: 2672843
- Ni YL, Kuan AS, Chen TY (2014) Activation and inhibition of TMEM16A calcium-activated chloride channels. PLoS One 9:e86734. doi:10.1371/journal.pone.0086734, PMID:24489780
- Ferrera L, Caputo A, Galietta L. (2010) TMEM16A protein: A new identity for Ca2+-dependent chloride channels. Physiology. 2010, DOI: 10.1152/physiol.00030.2010, PMID: 21186280