Anoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca 2+-activated cl − channel responsible for essential physiological processes. each monomer harbours a pore and a ca 2+-binding pocket; the voltage-dependent binding of two intracellular ca 2+ ions to the pocket gates the pore. However, in the absence of intracellular ca 2+ voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with ca 2+ for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/ Asp residues in the ca 2+-binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca 2+-binding pocket underlines TMEM16A activation in the absence of intracellular Ca 2+. Anoctamin-1 (ANO1 or TMEM16A) and Anoctamin-2 (ANO2 or TMEM16B) are the pore-forming subunits of Ca 2+-activated Cl − channels (CaCCs) 1-3. Several tissues express CaCCs that participate in vital physiological functions 4,5. Thus, a role for TMEM16A and TMEM16B in smooth muscle contraction, control of blood pressure, control of gastrointestinal movements, regulation of cardiac and neuronal excitability, fluid secretion in exocrine glands, secretion of melatonin, mucin and insulin, sperm capacitation and motility, inhibition of polyspermy, and sensory transduction was established using tissue-specific knockout mice 6-16. In addition, TMEM16A modulates the partitioning of membrane phosphoinositides and endocytic transport by controlling the [Cl − ] i 17. Overexpression of TMEM16A is associated with hypertension, increased cell proliferation and cancer progression 18-21. Activation of CaCCs is triggered by voltage-dependent binding of two Ca 2+ ions to the channel when the intracellular Ca 2+ concentration ([Ca 2+ ] i) increases 22-26. Structural and mutagenesis analysis show that Ca 2+ ions bind to an acidic Ca 2+ pocket formed by four Glu, one Asp and one Asn 25-27. The pocket is located near the cytosolic side facing the permeation pathway. However, other divalent cations and maybe trivalent cations too can support TMEM16A activation. Based on the cation concentrations to obtain the half-maximum response, the cation selectivity of TMEM16A gating machinery is Ca 2+ »Sr 2+ »Ba 2+ »Cd 2+ , 23,27-29. Gd 3+ may also activate TMEM16A since its application removed the inward rectification of the Gly644Pro TMEM16A mutant channel 30 .
Voltage-gated sodium channels (VGSCs) are heteromeric protein complexes that initiate action potentials in excitable cells. The voltage-gated sodium channel accessory subunit, Navβ1, allosterically modulates the α subunit pore structure upon binding. To date, the molecular determinants of the interface remain unknown. We made use of sequence, knowledge and structure-based methods to identify residues critical to the association of the α and β1 Nav1.4 subunits. The Navβ1 point mutant C43A disrupted the modulation of voltage dependence of activation and inactivation and delayed the peak current decay, the recovery from inactivation, and induced a use-dependent decay upon depolarisation at 1 Hz. The Navβ1 mutant R89A selectively delayed channel inactivation and recovery from inactivation and had no effect on voltage dependence or repetitive depolarisations. Navβ1 mutants Y32A and G33M selectively modified the half voltage of inactivation without altering the kinetics. Despite low sequence identity, highly conserved structural elements were identified. Our models were consistent with published data and may help relate pathologies associated with VGSCs to the Navβ1 subunit.
The molecular structure modeling of the β1 subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4) was carried out in the twilight zone of very low homology. Structural significance can per se be confounded with random sequence similarities. Hence, we combined (i) not automated computational modeling of weakly homologous 3D templates, some with interfaces to analogous structures to the pore-bearing Nav1.4 α subunit with (ii) site-directed mutagenesis (SDM), as well as (iii) electrophysiological experiments to study the structure and function of the β1 subunit. Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation) of Nav1.4 channels. This mutant type (T109A, N110A, herein called TANA) was expressed and tested on cells of hamster ovary (CHO). The present electrophysiological results showed that the double alanine substitution TANA disrupted channel inactivation as if the β1 subunit would not be in complex with the α subunit. Exhaustive and unbiased sampling of “all β proteins” (Ig-like, Ig) resulted in a plethora of 3D templates which were compared to the target secondary structure prediction. The location of TANA was made possible thanks to another “all β protein” structure in complex with an irreversible bound protein as well as a reversible protein–protein interface (our “Rosetta Stone” effect). This finding coincides with our electrophysiological data (disrupted β1-like voltage dependence) and it is safe to utter that the Nav1.4 α/β1 interface is likely to be of reversible nature.
The mechanism of inactivation of mammalian voltage-gated Na channels involves transient interactions between intracellular domains resulting in direct pore occlusion by the IFM motif and concomitant extracellular interactions with the β1 subunit. Naβ1 subunits constitute single-pass transmembrane proteins that form protein-protein associations with pore-forming α subunits to allosterically modulate the Na influx into the cell during the action potential of every excitable cell in vertebrates. Here, we explored the role of the intracellular IFM motif of rNa1.4 (skeletal muscle isoform of the rat Na channel) on the α-β1 functional interaction and showed for the first time that the modulation of β1 is independent of the IFM motif. We found that: (1) Na1.4 channels that lack the IFM inactivation particle can undergo a "C-type-like inactivation" albeit in an ultraslow gating mode; (2) β1 can significantly accelerate the inactivation of Na1.4 channels in the absence of the IFM motif. Previously, we identified two residues (T109 and N110) on the β1 subunit that disrupt the α-β1 allosteric modulation. We further characterized the electrophysiological effects of the double alanine substitution of these residues demonstrating that it decelerates inactivation and recovery from inactivation, abolishes the modulation of steady-state inactivation and induces a current rundown upon repetitive stimulation, thus causing a general loss of function. Our results contribute to delineating the process of the mammalian Na channel inactivation. These findings may be relevant to the design of pharmacological strategies, targeting β subunits to treat pathologies associated to Na current dysfunction.
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