TRPM5 is a transient Ca2+-activated cation channel responding to rapid changes in [Ca2+]i

Transient receptor potential (TRP) proteins are a diverse family of proteins with structural features typical of ion channels. TRPM5, a member of the TRPM subfamily, plays an important role in taste receptors, although its activation mechanism remains controversial and its function in signal transduction is unknown. Here we characterize the functional properties of heterologously expressed human TRPM5 in HEK-293 cells. TRPM5 displays characteristics of a calcium-activated, nonselective cation channel with a unitary conductance of 25 pS. TRPM5 is a monovalent-specific, nonselective cation channel that carries Na+, K+, and Cs+ ions equally well, but not Ca2+ ions. It is directly activated by [Ca2+]i at concentrations of 0.3-1 microM, whereas higher concentrations are inhibitory, resulting in a bell-shaped dose-response curve. It activates and deactivates rapidly even during sustained elevations in [Ca2+]i, thereby inducing a transient membrane depolarization. TRPM5 does not simply mirror levels of [Ca2+]i, but instead responds to the rate of change in [Ca2+]i in that it requires rapid changes in [Ca2+]i to generate significant whole-cell currents, whereas slow elevations in [Ca2+]i to equivalent levels are ineffective. Moreover, we demonstrate that TRPM5 is not limited to taste signal transduction, because we detect the presence of TRPM5 in a variety of tissues and we identify endogenous TRPM5-like currents in a pancreatic beta cell line. TRPM5 can be activated physiologically by inositol 1,4,5-trisphosphate-producing receptor agonists, and it may therefore couple intracellular Ca2+ release to electrical activity and subsequent cellular responses.     

Multiple Ca2+ channel types coexist to regulate synaptosomal neurotransmitter release.

The regulation of excitation-secretion coupling by Ca2+ channels is a fundamental property of the nerve terminal. Peptide toxins that block specific Ca2+ channel types have been used to identify which channels participate in neurotransmitter release. Subsecond measurements of [3H]-glutamate and [3H]dopamine release from rat striatal synaptosomes showed that P-type channels, which are sensitive to the Agelenopsis aperta venom peptide omega-Aga-IVA, trigger the release of both transmitters. Dopamine (but not glutamate) release was also controlled by N-type, omega-conotoxin-sensitive channels. With strong depolarizations, where neither toxin was very effective alone, a combination of omega-Aga-IVA and omega-conotoxin produced a synergistic inhibition of 60-80% of Ca(2+)-dependent dopamine release. The results suggest that multiple Ca2+ channel types coexist to regulate neurosecretion under normal physiological conditions in the majority of nerve terminals. P- and N-type channels coexist in dopaminergic terminals, while P-type and a omega-conotoxin- and omega-Aga-IVA-resistant channel coexist in glutamatergic terminals. Such an arrangement could lend a high degree of flexibility in the regulation of transmitter release under diverse conditions of stimulation and modulation.     

TRPM4, a Ca2+-activated nonselective cation channel in mouse sino-atrial node cells

OBJECTIVE: A calcium-activated nonselective cation channel (NSC(Ca)) has been recently described in several cardiac preparations. This channel is over-expressed in models of ventricular hypertrophy showing electrophysiological perturbations of heart activity, including occurrence of spontaneous activity. While these perturbations are currently attributed to a modification of the pacemaker I(f) current activity, arguments are also in favor of participation of an NSC(Ca). Similarly, the NSC(Ca) may be expressed in specialized pacemaker cells, i.e. sino-atrial node (SAN) cells. The aim of the present study was to detect such current in mouse pacemaker cells. METHODS: The inside-out configuration of the patch-clamp technique was used in freshly isolated SAN cells from adult mice. Also, RT-PCR and Western-blotting studies were used to probe for TRPM4 mRNA and protein expression. RESULTS: In these cells, an NSC(Ca) activity was detected. The channel is voltage dependant with a conductance of 20.9+/-0.5 pS (n = 11). It is equally permeable for Na+ and K+ but does not conduct Ca2+. It is activated by rise in intracellular calcium concentrations and blocked by intracellular ATP (0.5 mmol/L). Also, as a new property in cardiac cells, the channel is activated by internal application of phosphatidylinositol 4,5-bisphosphate (10 microM). It is reversibly inhibited by flufenamic acid and glibenclamide. This channel shows the hallmarks of the TRPM4 molecule, a member of the TRP melastatin subfamily. We confirm the expression of this TRP channel on SAN cells by Western blotting and RT-PCR and validate that TRPM4 is glibenclamide sensitive. CONCLUSION: TRPM4 is functionally expressed in SAN cells and may be a key player in the generation and/or perturbation of heart rhythm.     

Nicotine activates TRPM5-dependent and independent taste pathways

The orosensory responses elicited by nicotine are relevant for the development and maintenance of addiction to tobacco products. However, although nicotine is described as bitter tasting, the molecular and neural substrates encoding the taste of nicotine are unclear. Here, rats and mice were used to determine whether nicotine activates peripheral and central taste pathways via TRPM5-dependent mechanisms, which are essential for responses to other bitter tastants such as quinine, and/or via nicotinic acetylcholine receptors (nAChRs). When compared with wild-type mice, Trpm5^−/− mice had reduced, but not abolished, chorda tympani (CT) responses to nicotine. In both genotypes, lingual application of mecamylamine, a nAChR-antagonist, inhibited CT nerve responses to nicotine and reduced behavioral responses of aversion to this stimulus. In accordance with these findings, rats were shown to discriminate between nicotine and quinine presented at intensity-paired concentrations. Moreover, rat gustatory cortex (GC) neural ensemble activity could also discriminate between these two bitter tastants. Mecamylamine reduced both behavioral and GC neural discrimination between nicotine and quinine. In summary, nicotine elicits taste responses through peripheral TRPM5-dependent pathways, common to other bitter tastants, and nAChR-dependent and TRPM5-independent pathways, thus creating a unique sensory representation that contributes to the sensory experience of tobacco products.     

Pharmacological basis of Ca2+ channels and Ca2+ channel antagonists

Ca2+ plays multiple roles in muscle E-C coupling, secretion, and neural transmission, in addition to survival, proliferation, and death of cells. The voltage-dependent L-type Ca2+ channel is a transmembrane protein that selectively permeates Ca2+ on activation by membrane depolarization. Ca2+ channel blockers (or Ca2+ antagonists) selectively block this channel. The blocking action is exerted in a tissue-specific manner, which underlies the unique pharmacological properties of Ca2+ channel blockers. The later generation of slowly-acting and long-lasting Ca2+ channel blockers has been designed to overcome the side effects of classical Ca2+ channel blockers. The pharmacological and molecular basis for the unique action of Ca2+ channel blockers will be discussed.     

Structural studies of ion permeation and Ca2+ blockage of a bacterial channel mimicking the cyclic nucleotide-gated channel pore

Cyclic nucleotide-gated (CNG) channels play an essential role in the visual and olfactory sensory systems and are ubiquitous in eukaryotes. Details of their underlying ion selectivity properties are still not fully understood and are a matter of debate in the absence of high-resolution structures. To reveal the structural mechanism of ion selectivity in CNG channels, particularly their Ca(2+) blockage property, we engineered a set of mimics of CNG channel pores for both structural and functional analysis. The mimics faithfully represent the CNG channels they are modeled after, permeate Na(+) and K(+) equally well, and exhibit the same Ca(2+) blockage and permeation properties. Their high-resolution structures reveal a hitherto unseen selectivity filter architecture comprising three contiguous ion binding sites in which Na(+) and K(+) bind with different ion-ligand geometries. Our structural analysis reveals that the conserved acidic residue in the filter is essential for Ca(2+) binding but not through direct ion chelation as in the currently accepted view. Furthermore, structural insight from our CNG mimics allows us to pinpoint equivalent interactions in CNG channels through structure-based mutagenesis that have previously not been predicted using NaK or K(+) channel models.     

Ca2+-induced inhibition of the cardiac Ca2+ channel depends on calmodulin

Ca2+-induced inhibition of alpha1C voltage-gated Ca2+ channels is a physiologically important regulatory mechanism that shortens the mean open time of these otherwise long-lasting high-voltage-activated channels. The mechanism of action of Ca2+ has been a matter of some controversy, as previous studies have proposed the involvement of a putative Ca2+-binding EF hand in the C terminus of alpha1C and/or a sequence downstream from this EF-hand motif containing a putative calmodulin (CaM)-binding IQ motif. Previously, using site directed mutagenesis, we have shown that disruption of the EF-hand motif does not remove Ca2+ inhibition. We now show that the IQ motif binds CaM and that disruption of this binding activity prevents Ca2+ inhibition. We propose that Ca2+ entering through the voltage-gated pore binds to CaM and that the Ca/CaM complex is the mediator of Ca2+ inhibition.     

EF-hand protein Ca2+ buffers regulate Ca2+ influx and exocytosis in sensory hair cells

EF-hand Ca²⁺-binding proteins are thought to shape the spatiotemporal properties of cellular Ca²⁺ signaling and are prominently expressed in sensory hair cells in the ear. Here, we combined genetic disruption of parvalbumin-α, calbindin-D28k, and calretinin in mice with patch-clamp recording, in vivo physiology, and mathematical modeling to study their role in Ca²⁺ signaling, exocytosis, and sound encoding at the synapses of inner hair cells (IHCs). IHCs lacking all three proteins showed excessive exocytosis during prolonged depolarizations, despite enhanced Ca²⁺-dependent inactivation of their Ca²⁺ current. Exocytosis of readily releasable vesicles remained unchanged, in accordance with the estimated tight spatial coupling of Ca²⁺ channels and release sites (effective “coupling distance” of 17 nm). Substitution experiments with synthetic Ca²⁺ chelators indicated the presence of endogenous Ca²⁺ buffers equivalent to 1 mM synthetic Ca²⁺-binding sites, approximately half of them with kinetics as fast as 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Synaptic sound encoding was largely unaltered, suggesting that excess exocytosis occurs extrasynaptically. We conclude that EF-hand Ca²⁺ buffers regulate presynaptic IHC function for metabolically efficient sound coding.Intracellular Ca²⁺ signaling regulates a multitude of cellular processes. In sensory hair cells, Ca²⁺ is crucial for electrical frequency tuning, afferent synaptic transmission, and efferent modulation (reviewed in ref. 1). To separate these signaling pathways and maintain high temporal fidelity of neurotransmission, Ca²⁺ signals must be temporally limited and spatially confined to the site of action. Cells typically achieve this by localizing Ca²⁺ entry and by rapidly removing free Ca²⁺ ions via binding to cytosolic “buffers” and finally Ca²⁺ extrusion (2–4). Of the various EF-hand Ca²⁺-binding proteins, some seem to function primarily as Ca²⁺-dependent signaling proteins (e.g., calmodulin and Ca²⁺-binding proteins 1–8, CaBP1–8), whereas others [parvalbumin-α (PVα), calbindin-D28k (CB), and calretinin (CR)] are thought to mainly serve as mobile Ca²⁺ buffers.Hair cells of various species strongly express the Ca²⁺-binding proteins PV, CB, and, in some cases, CR. This possibly reflects the need for buffers with different biophysical properties to functionally isolate different Ca²⁺ signaling mechanisms, which are spatially not well separated in these compact epithelial cells. Ca²⁺-binding proteins are particularly abundant in frog and chicken hair cells, which contain millimolar concentrations of parvalbumin-3 (5) as well as of CR (6, 7). An immune-EM study in rats indicated hundreds of micromolar of proteinaceous Ca²⁺-binding sites in inner hair cells (IHCs) (8). A patch-clamp study in gerbil IHCs reported endogenous buffers equivalent to approximately 0.4 mM 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) (9). Genetic deletion of the EF-hand Ca²⁺-binding proteins PVα, CB, and CR in mice has facilitated the analysis of their function (10–13; reviewed in ref. 14), but the combined deletion of these proteins remains to be studied. IHCs provide an experimentally well-accessible presynaptic preparation that uses all three. Here, we studied IHC function and hearing in mice lacking the three buffers [triple buffer KO (TKO); Pv^−/−Cb^−/−Cr^−/−]. By using perforated and ruptured-patch recordings, we analyzed voltage-gated Ca²⁺ currents and exocytosis of Pv^−/−Cb^−/−Cr^−/− IHCs, in which we also substituted the deleted endogenous buffers with the synthetic Ca²⁺ chelators EGTA or BAPTA. Auditory systems function was probed by measuring otoacoustic emissions and auditory brainstem responses (ABRs) as well as by recordings from single spiral ganglion neurons (SGNs). We performed mathematical modeling to estimate concentrations of the endogenous mobile Ca²⁺ buffers and to better understand how these proteins control exocytosis at IHC synapses. We conclude that the endogenous buffer capacity of IHCs is well approximated by 1 mM synthetic Ca²⁺-binding sites with different kinetics. A tight spatial coupling between Ca²⁺ channels and sensors of exocytosis (Ca²⁺ channel-exocytosis coupling) precludes interference of PVα, CB, and CR with fusion of the readily releasable pool of vesicles (RRP). Instead, we suggest that these buffers jointly regulate IHC presynaptic function by restricting neurotransmitter release to active zones (AZs).     

Visualization of Ca2+-induced phospholipid domains.

Large vesicles (5-15 microns) were formed by hydrating a dried lipid film containing phospholipids labeled with a fluorophore in one fatty acid chain. By using a fluorescence microscope attached to a low-light-intensity charge-coupled-device camera and digital-image processor, the vesicles were easily viewed and initially showed uniform fluorescence intensity across the surface. The fluorescence pattern of vesicles made with a fluorophore attached to phosphatidylcholine or phosphatidylethanolamine was unaffected by the presence of divalent cations such as Ca2+, Mg2+, Mn2+, Zn2+, or Cd2+. The fluorescence pattern of vesicles containing a fluorophore attached to the acidic phospholipids phosphatidylserine or phosphatidic acid showed distinct differences when treated with Ca2+ or Cd2+, although they were unaffected by Mg2+, Mn2+, or Zn2+. Treatment with 2.0 mM Ca2+ or Cd2+ resulted in the movement of the fluorophore to a single large patch on the surface of the vesicle. When vesicles were formed in the presence of 33 mol % cholesterol, patching was seen at a slightly lower Ca2+ concentration (1.0 mM). The possibility of interactions between Ca2+ and acidic phospholipids in plasma membranes was investigated by labeling erythrocytes and erythrocyte ghosts with fluorescent phosphatidic acid. When Ca2+ was added, multiple (five or six) small patches were seen per individual cell. The same pattern was observed when vesicles formed from whole lipid extracts of erythrocytes were labeled with fluorescent phosphatidic acid and then treated with Ca2+. This shows that the size and distribution of the Ca2+-induced domains depend on phospholipid composition.     

Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight

Neuronal Ca²⁺ signals can affect excitability and neural circuit formation. Ca²⁺ signals are modified by Ca²⁺ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca²⁺ stores and their release to neuronal processes is poorly understood. Here, we show by neuron-specific siRNA depletion that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca²⁺ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP₃R) can partially compensate for their loss of flight. Ca²⁺ measurements show that Orai gain-of-function contributes to the quanta of Ca²⁺-release through mutant InsP₃Rs and elevates store-operated Ca²⁺ entry in Drosophila neurons. Our data show that replenishment of intracellular store Ca²⁺ in neurons is required for Drosophila flight.     

Ca2+ channel subtypes and pharmacology in the kidney

A large body of evidence has accrued indicating that voltage-gated Ca(2+) channel subtypes, including L-, T-, N-, and P/Q-type, are present within renal vascular and tubular tissues, and the blockade of these Ca(2+) channels produces diverse actions on renal microcirculation. Because nifedipine acts exclusively on L-type Ca(2+) channels, the observation that nifedipine predominantly dilates afferent arterioles implicates intrarenal heterogeneity in the distribution of L-type Ca(2+) channels and suggests that it potentially causes glomerular hypertension. In contrast, recently developed Ca(2+) channel blockers (CCBs), including mibefradil and efonidipine, exert blocking action on L-type and T-type Ca(2+) channels and elicit vasodilation of afferent and efferent arterioles, which suggests the presence of T-type Ca(2+) channels in both arterioles and the distinct impact on intraglomerular pressure. Recently, aldosterone has been established as an aggravating factor in kidney disease, and T-type Ca(2+) channels mediate aldosterone release as well as its effect on renal efferent arteriolar tone. Furthermore, T-type CCBs are reported to exert inhibitory action on inflammatory process and renin secretion. Similarly, N-type Ca(2+) channels are present in nerve terminals, and the inhibition of neurotransmitter release by N-type CCBs (eg, cilnidipine) elicits dilation of afferent and efferent arterioles and reduces glomerular pressure. Collectively, the kidney is endowed with a variety of Ca(2+) channel subtypes, and the inhibition of these channels by their specific CCBs leads to variable impact on renal microcirculation. Furthermore, multifaceted activity of CCBs on T- and N-type Ca(2+) channels may offer additive benefits through nonhemodynamic mechanisms in the progression of chronic kidney disease.     

Receptor-mediated activation of a plant Ca2+-permeable ion channel involved in pathogen defense

Pathogen recognition at the plant cell surface typically results in the initiation of a multicomponent defense response. Transient influx of Ca2+ across the plasma membrane is postulated to be part of the signaling chain leading to pathogen resistance. Patch-clamp analysis of parsley protoplasts revealed a novel Ca2+-permeable, La3+-sensitive plasma membrane ion channel of large conductance (309 pS in 240 mM CaCl2). At an extracellular Ca2+ concentration of 1 mM, which is representative of the plant cell apoplast, unitary channel conductance was determined to be 80 pS. This ion channel (LEAC, for large conductance elicitor-activated ion channel) is reversibly activated upon treatment of parsley protoplasts with an oligopeptide elicitor derived from a cell wall protein of Phytophthora sojae. Structural features of the elicitor found previously to be essential for receptor binding, induction of defense-related gene expression, and phytoalexin formation are identical to those required for activation of LEAC. Thus, receptor-mediated stimulation of this channel appears to be causally involved in the signaling cascade triggering pathogen defense in parsley.     

Cardiac function involving the T-type Ca2+ channel

Functional involvement of the T-type calcium channel in the heart excitation at the pathophysiological conditions has been elucidated. The T-type channel is classified as the low voltage-activated channel (LVA) solely in the voltage activated calcium channel. In 1998, Perez-Reyes and coworkers cloned the first LVA alpha(1) subunit, which was named as alpha(1G) or Ca(V)3.1. In the cardiac muscle, alpha(1G) and the other clone alpha(1H) are dominantly expressed in the sinoatrial node, atrioventricular node and other signal conduction tissues. Abnormal activity of T channels has been suggested in the following cardiovascular diseases: hypertension, cardiac hypertrophy, cardiac infarction. The cloning of the T-type calcium channel allows us to understand the function of the channel in detail and options for therapeutics in the T-type channel-related diseases.