Recognition of sweet, bitter and umami tastes requires the non-vesicular release from taste bud cells of adenosine 5′-triphosphate (ATP), which acts as a neurotransmitter to activate afferent neural gustatory pathways1. However, how ATP is released to fulfill this function is not fully understood. Here we show that calcium homeostasis modulator 1 (CALHM1), a voltage-gated ion channel2,3, is indispensable for taste stimuli-evoked ATP release from sweet-, bitter- and umami-sensing taste bud cells. Calhm1 knockout mice have severely impaired perceptions of sweet, bitter and umami compounds, whereas sour and salty taste recognition remains mostly normal. Calhm1 deficiency affects taste perception without interfering with taste cell development or integrity. CALHM1 is expressed specifically in sweet/bitter/umami-sensing type II taste bud cells. Its heterologous expression induces a novel ATP permeability that releases ATP from cells in response to manipulations that activate the CALHM1 ion channel. Knockout of Calhm1 strongly reduces voltage-gated currents in type II cells and taste-evoked ATP release from taste buds without affecting the excitability of taste cells to taste stimuli. Thus, CALHM1 is a voltage-gated ATP release channel required for sweet, bitter and umami taste perception.

The activation of large conductance Ca2+-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K+ (KV) channels. Yet BK and KV channels share many conserved charged residues in transmembrane segments S1–S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (Po) and IK kinetics (τ(IK)) over an extended voltage range in 0–50 μM [Ca2+]i. mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of PO. The voltage dependence of PO and τ(IK) at extreme negative potentials was also reduced, implying that the closed–open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and KV channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to KV channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1–S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3–7 kcal mol−1, indicating a strong contribution of non–voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.

Alzheimer's disease | mutagenesis | selectivity | neurodegeneration | polymorphism O riginally identified as a possible modifier of the age of onset of Alzheimer's disease (1, 2), calcium homeostasis modulator 1 (CALHM1) encodes a glycosylated membrane protein expressed throughout the brain that lacks homology to other proteins. Six human CALHM homologs have been identified, with alternatively spliced variants and different expression patterns throughout the body, and CALHM1 is conserved across >20 species. Expression of recombinant human CALHM1 in mammalian cells was found to strongly influence processing of amyloid precursor protein to amyloid beta (Aβ) under an experimental protocol that involved removal of Ca 2+ o for several minutes and its subsequent restoration to the bathing medium (1). This procedure resulted in a large rise of [Ca 2+ ] i . Accordingly, it was speculated that CALHM1 influences Aβ production by altering cellular Ca 2+ homeostasis. CALHM1 was found to homo-multimerize and it was speculated that it might function as an ion channel component or regulator of membrane ion conductances (1 Results CALHM1 Expression Induces a Voltage-Dependent Plasma MembraneConductance. Previously, outwardly rectified ion currents were observed in CALHM1-expressing Xenopus oocytes and CHO cells by a slow voltage ramp protocol (1). However, those experiments did not establish whether CALHM1 is an essential component of an underlying ion channel(s), accessory protein, or actual pore-forming subunit. Furthermore the detailed permeation and gating properties of the conductance were not determined. In addition, precautions were not taken to fully ensure lack of contribution of endogenous conductances. To distinguish whether CALHM1 is a unique ion channel or a regulator of endogenous channels, plasma membrane currents were recorded in Xenopus oocytes under conditions that minimized contributions of endogenous conductances (Fig. S1) (3, 4). Membrane depolarization in solutions containing 2 mM Ca 2+ and 1 mM Mg 2+ generated large outward currents that activated slowly (τ ∼ 3.11 ± 0.28 s at +60 mV; n = 10) and deactivated at hyperpolarized voltages (τ = 0.204 ± 0.022 s at −80 mV; n = 10) specifically in CALHM1-expressing oocytes (Fig. 1A). Expression of CALHM1-EGFP localized to the plasma membrane (Fig. 1B). Similar results were obtained in transiently transfected N2A mammalian neuroblastoma cells (Fig. 1C). Thus, expression of CALHM1 induced a voltage-dependent plasma membrane conductance.The monovalent ion permeabilities of this conductance were estimated by changing-bath [NaCl] in a nominally 0-Ca 2+ solution (free [Ca 2+ ] ∼10 μM) and measuring shifts of reversal potential, ΔE rev (Fig. 1D). Using the Goldman-Hodgkin-Katz (GHK) constant field equation, the relative permeabilities were estimated as P Na :P K :P Cl = 1:1.17:0.56. Similar results were obtained with bath Na + replaced by K + in either 0 or 2 mM Ca 2+ o .

Binding of sweet, umami, and bitter tastants to G protein-coupled receptors (GPCRs) in apical membranes of type II taste bud cells (TBCs) triggers action potentials that activate a voltage-gated nonselective ion channel to release ATP to gustatory nerves mediating taste perception. Although calcium homeostasis modulator 1 (CALHM1) is necessary for ATP release, the molecular identification of the channel complex that provides the conductive ATP-release mechanism suitable for action potential-dependent neurotransmission remains to be determined. Here we show that CALHM3 interacts with CALHM1 as a pore-forming subunit in a CALHM1/CALHM3 hexameric channel, endowing it with fast voltage-activated gating identical to that of the ATP-release channel in vivo. Calhm3 is co-expressed with Calhm1 exclusively in type II TBCs, and its genetic deletion abolishes taste-evoked ATP release from taste buds and GPCR-mediated taste perception. Thus, CALHM3, together with CALHM1, is essential to form the fast voltage-gated ATP-release channel in type II TBCs required for GPCR-mediated tastes.

Background: CALHM1 is an ion channel for which structural information is lacking. Results: CALHM1 has poor ion selectivity and a wide (ϳ14 Å) pore and is a hexamer, with monomers having four transmembrane domains with cytoplasmic termini. Conclusion: CALHM1 shares structural features with pannexins, connexins, and innexins. Significance: CALHMs, connexins, and pannexins/innexins are three structurally related protein families with shared and distinct functional properties.

SUMMARY The mitochondrial calcium uniporter complex is essential for calcium (Ca2+) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca2+ signal transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-molecule modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate-and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-positive hits, we identify mitoxantrone out of more than 600 clinically approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compounds that target mitochondrial functions.

BK (Slo1) potassium channels are activated by millimolar intracellular Mg2+ as well as micromolar Ca2+ and membrane depolarization. Mg2+ and Ca2+ act in an approximately additive manner at different binding sites to shift the conductance–voltage (GK-V) relation, suggesting that these ligands might work through functionally similar but independent mechanisms. However, we find that the mechanism of Mg2+ action is highly dependent on voltage sensor activation and therefore differs fundamentally from that of Ca2+. Evidence that Ca2+ acts independently of voltage sensor activation includes an ability to increase open probability (PO) at extreme negative voltages where voltage sensors are in the resting state; 2 μM Ca2+ increases PO more than 15-fold at −120 mV. However 10 mM Mg2+, which has an effect on the GK-V relation similar to 2 μM Ca2+, has no detectable effect on PO when voltage sensors are in the resting state. Gating currents are only slightly altered by Mg2+ when channels are closed, indicating that Mg2+ does not act merely to promote voltage sensor activation. Indeed, channel opening is facilitated in a voltage-independent manner by Mg2+ in a mutant (R210C) whose voltage sensors are constitutively activated. Thus, 10 mM Mg2+ increases PO only when voltage sensors are activated, effectively strengthening the allosteric coupling of voltage sensor activation to channel opening. Increasing Mg2+ from 10 to 100 mM, to occupy very low affinity binding sites, has additional effects on gating that more closely resemble those of Ca2+. The effects of Mg2+ on steady-state activation and IK kinetics are discussed in terms of an allosteric gating scheme and the state-dependent interactions between Mg2+ and voltage sensor that may underlie this mechanism.

Calcium homeostasis modulator 1 (CALHM1), formerly known as FAM26C, was recently identified as a physiologically important plasma membrane ion channel. CALHM1 and its Caenorhabditis elegans homolog, CLHM-1, are regulated by membrane voltage and extracellular Ca2+ concentration ([Ca2+]o). In the presence of physiological [Ca2+]o (~1.5 mM), CALHM1 and CLHM-1 are closed at resting membrane potentials but can be opened by strong de-polarizations. Reducing [Ca2+]o increases channel open probability, enabling channel activation at negative membrane potentials. Together, voltage and Ca2+o allosterically regulate CALHM channel gating. Through convergent evolution, CALHM has structural features that are reminiscent of connexins and pannexins/innexins/LRRC8 (volume-regulated anion channel (VRAC)) gene families, including four trans-membrane helices with cytoplasmic amino and carboxyl termini. A CALHM1 channel is a hexamer of CALHM1 monomers with a functional pore diameter of ~14 Å. CALHM channels discriminate poorly among cations and anions, with signaling molecules including Ca2+ and ATP able to permeate through its pore. CALHM1 is expressed in the brain where it plays an important role in cortical neuron excitability induced by low [Ca2+]o and in type II taste bud cells in the tongue that sense sweet, bitter, and umami tastes where it functions as an essential ATP release channel to mediate nonsynaptic neuro-transmitter release. CLHM-1 is expressed in C. elegans sensory neurons and body wall muscles, and its genetic deletion causes locomotion defects. Thus, CALHM is a voltage- and Ca2+o-gated ion channel, permeable to large cations and anions, that plays important roles in physiology.