Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeation of the T-type Ca(2+) channel alpha(1G)

The structural determinant of the permeation and selectivity properties of high voltage-activated (HVA) Ca(2+) channels is a locus formed by four glutamate residues (EEEE), one in each P-region of the domains I-IV of the alpha(1) subunit. We tested whether the divergent aspartate residues of the EEDD locus of low voltage-activated (LVA or T-type) Ca(2+) channels account for the distinctive permeation and selectivity features of these channels. Using the whole-cell patch-clamp technique in the HEK293 expression system, we studied the properties of the alpha(1G) T-type, the alpha(1C) L-type Ca(2+) channel subunits, and alpha(1G) pore mutants, containing aspartate-to-glutamate conversions in domain III, domain IV, or both. Three characteristic features of HVA Ca(2+) channel permeation, i.e. (a) Ba(2+) over Ca(2+) permeability, (b) Ca(2+)/Ba(2+) anomalous mole fraction effect (AMFE), and (c) high Cd(2+) sensitivity, were conferred on the domain III mutant (EEED) of alpha(1G). In contrast, the relative Ca(2+)/Ba(2+) permeability and the lack of AMFE of the alpha(1G) wild type channel were retained in the domain IV mutant (EEDE). The double mutant (EEEE) displayed AMFE and a Cd(2+) sensitivity similar to that of alpha(1C), but currents were larger in Ca(2+)- than in Ba(2+)-containing solutions. The mutation in domain III, but not that in domain IV, consistently displayed outward fluxes of monovalent cations. H(+) blocked Ca(2+) currents in all mutants more efficiently than in alpha(1G). In addition, activation curves of all mutants were displaced to more positive voltages and had a larger slope factor than in alpha(1G) wild type. We conclude that the aspartate residues of the EEDD locus of the alpha(1G) Ca(2+) channel subunit not only control its permeation properties, but also affect its activation curve. The mutation of both divergent aspartates only partially confers HVA channel permeation properties to the alpha(1G) Ca(2+) channel subunit.     

Biophysics and structure-function relationship of T-type Ca2+ channels

T-type channels are distinguished among voltage-gated Ca2+ channels by their low voltage thresholds for activation and inactivation, fast inactivation and small single channel conductance in isotonic Ba2+. Detailed biophysical and pharmacological characterization of native T-type channels indicated that these channels represent a heterogeneous family. Cloning of three family members (CaV3.1-3.3) confirmed these observations and allowed the study of the structure-function relationship of these channels. T-type channels are likely heterotetrameric structures consisting of a single polypeptide of four homologous domains (I-IV), each one containing six transmembrane spans (S1-S6), and cytoplasmic N- and C-termini. Structure-function studies have revealed that fast macroscopic inactivation of CaV3.1 is modulated by specific residues in the proximal C-terminus and in the transmembrane domain IIIS6. The particular gating properties within the T-type channel subfamily are determined by several parts of the protein, whereas differences with respect to high-voltage-activated Ca2+ channels are mostly determined by domains I, II and III. Several gating properties are affected by alternative splicing, C-terminal truncations and mutations associated to idiopathic epilepsy. Intriguingly, the aspartate residues of the EEDD locus of the selectivity filter not only determine the permeation properties and the block by Cd2+ and protons, but also activation and deactivation. Mutagenesis has also revealed that the outermost arginines of the S4 segment of domain IV influence the activation of CaV3.2, though no specific voltage-sensing amino acid has yet been properly identified. The selective modulation of CaV3.2 by G-proteins, CaMKII and PKA is determined by the II-III linker and the high-affinity inhibition of CaV3.2 by Ni2+ relies on a histidine residue in the IS3-S4 linker. Certainly, more structure-function studies are needed for a better understanding of T-type channel physiology and the rational design of treatments against T-type channel-related pathologies.     

Extracellular Ca2+ modulates the effects of protons on gating and conduction properties of the T-type Ca2+ channel alpha1G (CaV3.1)

Since Ca2+ is a major competitor of protons for the modulation of high voltage-activated Ca2+ channels, we have studied the modulation by extracellular Ca2+ of the effects of proton on the T-type Ca2+ channel alpha1G (CaV3.1) expressed in HEK293 cells. At 2 mM extracellular Ca2+ concentration, extracellular acidification in the pH range from 9.1 to 6.2 induced a positive shift of the activation curve and increased its slope factor. Both effects were significantly reduced if the concentration was increased to 20 mM or enhanced in the absence of Ca2+. Extracellular protons shifted the voltage dependence of the time constant of activation and decreased its voltage sensitivity, which excludes a voltage-dependent open pore block by protons as the mechanism modifying the activation curve. Changes in the extracellular pH altered the voltage dependence of steady-state inactivation and deactivation kinetics in a Ca2+-dependent manner, but these effects were not strictly correlated with those on activation. Model simulations suggest that protons interact with intermediate closed states in the activation pathway, decreasing the gating charge and shifting the equilibrium between these states to less negative potentials, with these effects being inhibited by extracellular Ca2+. Extracellular acidification also induced an open pore block and a shift in selectivity toward monovalent cations, which were both modulated by extracellular Ca2+ and Na+. Mutation of the EEDD pore locus altered the Ca2+-dependent proton effects on channel selectivity and permeation. We conclude that Ca2+ modulates T-type channel function by competing with protons for binding to surface charges, by counteracting a proton-induced modification of channel activation and by competing with protons for binding to the selectivity filter of the channel.     

CaV3.1 channel pore pseudo-symmetry revealed by selectivity filter mutations in its domains I/II

There is growing evidence indicating that the pore structure of voltage-gated ion channels (VGICs) influences gating besides their conductance. Regarding low voltage-activated (LVA) Ca²⁺ channels, it has been demonstrated that substitutions of the pore aspartate (D) by a glutamate (D-to-E substitution) in domains III and IV alter channel gating properties such as a positive shift in the channel activation voltage dependence. In the present report, we evaluated the effects of E-to-D substitution in domains I and II on the Ca_V3.1 channel gating properties. Our results indicate that substitutions in these two domains differentially modify the gating properties of Ca_V3.1 channels. The channel with a single mutation in domain I (DEDD) presented slower activation and faster inactivation kinetics and a slower recovery from inactivation, as compared with the WT channel. In contrast, the single mutant in domain II (EDDD) presented a small but significant negative shift of activation voltage dependence with faster activation and slower inactivation kinetics. Finally, the double mutant channel (DDDD) presented somehow intermediate properties with respect to the two single mutants but with fastest deactivation kinetics. Overall, our results indicate that single amino acid modification of the selectivity filter of LVA Ca²⁺ channels in distinct domains differentially influence their gating properties, supporting a pore pseudo-symmetry.     

A naturally occurring amino acid substitution in the voltage-dependent sodium channel selectivity filter affects channel gating

The pore of sodium channels contains a selectivity filter made of 4 amino acids, D/E/K/A. In voltage sensitive sodium channel (Nav) channels from jellyfish to human the fourth amino acid is Ala. This Ala, when mutated to Asp, promotes slow inactivation. In some Nav channels of pufferfishes, the Ala is replaced with Gly. We studied the biophysical properties of an Ala-to-Gly substitution (A1529G) in rat Nav1.4 channel expressed in Xenopus oocytes alone or with a β1 subunit. The Ala-to-Gly substitution does not affect monovalent cation selectivity and positively shifts the voltage-dependent inactivation curve, although co-expression with a β1 subunit eliminates the difference between A1529G and WT. There is almost no difference in channel fast inactivation, but the β1 subunit accelerates WT current inactivation significantly more than it does the A1529G channels. The Ala-to-Gly substitution mainly influences the rate of recovery from slow inactivation. Again, the β1 subunit is less effective on speeding recovery of A1529G than the WT. We searched Nav channels in numerous databases and noted at least four other independent Ala-to-Gly substitutions in Nav channels in teleost fishes. Thus, the Ala-to-Gly substitution occurs more frequently than previously realized, possibly under selection for alterations of channel gating.     

Molecular determinant for specific Ca/Ba selectivity profiles of low and high threshold Ca2+ channels

Voltage-gated Ca(2+) channels (VGCC) play a key role in many physiological functions by their high selectivity for Ca(2+) over other divalent and monovalent cations in physiological situations. Divalent/monovalent selection is shared by all VGCC and is satisfactorily explained by the existence, within the pore, of a set of four conserved glutamate/aspartate residues (EEEE locus) coordinating Ca(2+) ions. This locus however does not explain either the choice of Ca(2+) among other divalent cations or the specific conductances encountered in the different VGCC. Our systematic analysis of high- and low-threshold VGCC currents in the presence of Ca(2+) and Ba(2+) reveals highly specific selectivity profiles. Sequence analysis, molecular modeling, and mutational studies identify a set of nonconserved charged residues responsible for these profiles. In HVA (high voltage activated) channels, mutations of this set modify divalent cation selectivity and channel conductance without change in divalent/monovalent selection, activation, inactivation, and kinetics properties. The Ca(V)2.1 selectivity profile is transferred to Ca(V)2.3 when exchanging their residues at this location. Numerical simulations suggest modification in an external Ca(2+) binding site in the channel pore directly involved in the choice of Ca(2+), among other divalent physiological cations, as the main permeant cation for VGCC. In LVA (low voltage activated) channels, this locus (called DCS for divalent cation selectivity) also influences divalent cation selection, but our results suggest the existence of additional determinants to fully recapitulate all the differences encountered among LVA channels. These data therefore attribute to the DCS a unique role in the specific shaping of the Ca(2+) influx between the different HVA channels.     

pH modification of human T-type calcium channel gating

External pH (pH(o)) modifies T-type calcium channel gating and permeation properties. The mechanisms of T-type channel modulation by pH remain unclear because native currents are small and are contaminated with L-type calcium currents. Heterologous expression of the human cloned T-type channel, alpha1H, enables us to determine the effect of changing pH on isolated T-type calcium currents. External acidification from pH(o) 8.2 to pH(o) 5.5 shifts the midpoint potential (V(1/2)) for steady-state inactivation by 11 mV, shifts the V(1/2) for maximal activation by 40 mV, and reduces the voltage dependence of channel activation. The alpha1H reversal potential (E(rev)) shifts from +49 mV at pH(o) 8.2 to +36 mV at pH(o) 5.5. The maximal macroscopic conductance (G(max)) of alpha1H increases at pH(o) 5.5 compared to pH(o) 8.2. The E(rev) and G(max) data taken together suggest that external protons decrease calcium/monovalent ion relative permeability. In response to a sustained depolarization alpha1H currents inactivate with a single exponential function. The macroscopic inactivation time constant is a steep function of voltage for potentials < -30 mV at pH(o) 8.2. At pH(o) 5.5 the voltage dependence of tau(inact) shifts more depolarized, and is also a more gradual function of voltage. The macroscopic deactivation time constant (tau(deact)) is a function of voltage at the potentials tested. At pH(o) 5.5 the voltage dependence of tau(deact) is simply transposed by approximately 40 mV, without a concomitant change in the voltage dependence. Similarly, the delay in recovery from inactivation at V(rec) of -80 mV in pH(o) 5.5 is similar to that with a V(rec) of -120 mV at pH(o) 8.2. We conclude that alpha1H is uniquely modified by pH(o) compared to other calcium channels. Protons do not block alpha1H current. Rather, a proton-induced change in activation gating accounts for most of the change in current magnitude with acidification.     

Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions

Inactivation is an intrinsic property of numerous voltage-gated K⁺ (Kv) channels and can occur by N-type or/and C-type mechanisms. N-type inactivation is a fast, voltage independent process, coupled to activation, with each inactivation particle of a tetrameric channel acting independently. In N-type inactivation, a single inactivation particle is necessary and sufficient to occlude the pore. C-type inactivation is a slower process, involving the outermost region of the pore and is mediated by a concerted, highly cooperative interaction between all four subunits. Inactivation of Kv7.1 channels does not exhibit the hallmarks of N- and C-type inactivation. Inactivation of WT Kv7.1 channels can be revealed by hooked tail currents that reflects the recovery from a fast and voltage-independent inactivation process. However, several Kv7.1 mutants such as the pore mutant L273F generate an additional voltage-dependent slow inactivation. The subunit interactions during this slow inactivation gating remain unexplored. The goal of the present study was to study the nature of subunit interactions along Kv7.1 inactivation gating, using concatenated tetrameric Kv7.1 channel and introducing sequentially into each of the four subunits the slow inactivating pore mutation L273F. Incorporating an incremental number of inactivating mutant subunits did not affect the inactivation kinetics but slowed down the recovery kinetics from inactivation. Results indicate that Kv7.1 inactivation gating is not compatible with a concerted cooperative process. Instead, adding an inactivating subunit L273F into the Kv7.1 tetramer incrementally stabilizes the inactivated state, which suggests that like for activation gating, Kv7.1 slow inactivation gating is not a concerted process.     

Mechanism of arachidonic acid modulation of the T-type Ca2+ channel alpha1G

Arachidonic acid (AA) modulates T-type Ca(2+) channels and is therefore a potential regulator of diverse cell functions, including neuronal and cardiac excitability. The underlying mechanism of modulation is unknown. Here we analyze the effects of AA on the T-type Ca(2+) channel alpha(1G) heterologously expressed in HEK-293 cells. AA inhibited alpha(1G) currents within a few minutes, regardless of preceding exposure to inhibitors of AA metabolism (ETYA and 17-ODYA). Current inhibition was also observed in cell-free inside-out patches, indicating a membrane-delimited interaction of AA with the channel. AA action was consistent with a decrease of the open probability without changes in the size of unitary currents. AA shifted the inactivation curve to more negative potentials, increased the speed of macroscopic inactivation, and decreased the extent of recovery from inactivation at -80 mV but not at -110 mV. AA induced a slight increase of activation near the threshold and did not significantly change the deactivation kinetics or the rectification pattern. We observed a tonic current inhibition, regardless of whether the channels were held in resting or inactivated states during AA perfusion, suggesting a state-independent interaction with the channel. Model simulations indicate that AA inhibits T-type currents by switching the channels into a nonavailable conformation and by affecting transitions between inactivated states, which results in the negative shift of the inactivation curve. Slow-inactivating alpha(1G) mutants showed an increased affinity for AA with respect to the wild type, indicating that the structural determinants of fast inactivation are involved in the AA-channel interaction.     

Independent and joint modulation of rat Nav1.6 voltage-gated sodium channels by coexpression with the auxiliary β1 and β2 subunits

The Na_v1.6 voltage-gated sodium channel α subunit isoform is the most abundant isoform in the brain and is implicated in the transmission of high frequency action potentials. Purification and immunocytochemical studies imply that Na_v1.6 exist predominantly as Na_v1.6 + β1 + β2 heterotrimeric complexes. We assessed the independent and joint effects of the rat β1 and β2 subunits on the gating and kinetic properties of rat Na_v1.6 channels by recording whole-cell currents in the two-electrode voltage clamp configuration following transient expression in Xenopus oocytes. The β1 subunit accelerated fast inactivation of sodium currents but had no effect on the voltage dependence of their activation and steady-state inactivation and also prevented the decline of currents following trains of high-frequency depolarizing prepulses. The β2 subunit selectively retarded the fast phase of fast inactivation and shifted the voltage dependence of activation towards depolarization without affecting other gating properties and had no effect on the decline of currents following repeated depolarization. The β1 and β2 subunits expressed together accelerated both kinetic phases of fast inactivation, shifted the voltage dependence of activation towards hyperpolarization, and gave currents with a persistent component typical of those recorded from neurons expressing Na_v1.6 sodium channels. These results identify unique effects of the β1 and β2 subunits and demonstrate that joint modulation by both auxiliary subunits gives channel properties that are not predicted by the effects of individual subunits.     

A point mutation in the pore region alters gating, Ca(2+) blockage, and permeation of olfactory cyclic nucleotide-gated channels

Upon stimulation by odorants, Ca(2+) and Na(+) enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide-gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the alpha subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide-gated channels play a similar role. We studied the corresponding glutamate (E340) of the alpha subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca(2+) and Mg(2+). E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca(2+) and Mg(2+) powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca(2+) relative to Na(+) was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca(2+) and Mg(2+), and affects channel gating by cAMP more than by cGMP.     

Sodium channels: ionic model of slow inactivation and state-dependent drug binding

Inactivation is a fundamental property of voltage-gated ion channels. Fast inactivation of Na(+) channels involves channel block by the III-IV cytoplasmic interdomain linker. The mechanisms of nonfast types of inactivation (intermediate, slow, and ultraslow) are unclear, although the ionic environment and P-loops rearrangement appear to be involved. In this study, we employed a TTX-based P-loop domain model of a sodium channel and the MCM method to investigate a possible role of P-loop rearrangement in the nonfast inactivation. Our modeling predicts that Na(+) ions can bind between neighboring domains in the outer-carboxylates ring EEDD, forming an ordered structure with interdomain contacts that stabilize the conducting conformation of the outer pore. In this model, the permeant ions can transit between the EEDD ring and the selectivity filter ring DEKA, retaining contacts with at least two carboxylates. In the absence of Na(+), the electrostatic repulsion between the EEDD carboxylates disrupts the permeable configuration. In this Na(+)-deficient model, the region between the EEDD and DEKA rings is inaccessible for Na(+) but is accessible for TMA. Taken together, these results suggest that Na(+)-saturated models are consistent with experimental characteristics of the open channels, whereas Na(+)-deficient models are consistent with experimentally defined properties of the slow-inactivated channels. Our calculations further predict that binding of LAs to the inner pore would depend on whether Na(+) occupies the DEKA ring. In the absence of Na(+) in the DEKA ring, the cationic group of lidocaine occurs in the focus of the pore helices' macrodipoles and would prevent occupation of the ring by Na(+). Loading the DEKA ring with Na(+) results in the electrostatic repulsion with lidocaine. Thus, there are antagonistic relations between a cationic ligand bound in the inner pore and Na(+) in the DEKA ring.     

Specific properties of T-type calcium channels generated by the human alpha 1I subunit

We have cloned and expressed a human alpha(1I) subunit that encodes a subtype of T-type calcium channels. The predicted protein is 95% homologous to its rat counterpart but has a distinct COOH-terminal region. Its mRNA is detected almost exclusively in the human brain, as well as in adrenal and thyroid glands. Calcium currents generated by the functional expression of human alpha(1I) and alpha(1G) subunits in HEK-293 cells were compared. The alpha(1I) current activated and inactivated approximately 10 mV more positively. Activation and inactivation kinetics were up to six times slower, while deactivation kinetics was faster and showed little voltage dependence. A slower recovery from inactivation, a lower sensitivity to Ni(2+) ions (IC(50) approximately 180 micrometer), and a larger channel conductance (approximately 11 picosiemens) were the other discriminative features of the alpha(1I) current. These data demonstrate that the alpha(1I) subunit encodes T-type Ca(2+) channels functionally distinct from those generated by the human alpha(1G) or alpha(1H) subunits and point out that human and rat alpha(1I) subunits have species-specific properties not only in their primary sequence, but also in their expression profile and electrophysiological behavior.     

T-type calcium channels and tumor proliferation

The role of T-type Ca2+ channels in proliferation of tumor cells is reviewed. Intracellular Ca2+ is important in controlling proliferation as evidenced by pulses, or oscillations, of intracellular Ca2+ which occur in a cell cycle-dependent manner in many tumor cells. Voltage-gated calcium channels, such as the T-type Ca2+ channel, are well suited to participate in such oscillations due to their unique activation/inactivation properties. Expression of the T-type Ca2+ channels has been reported in numerous types of tumors, and has been shown to be cell cycle-dependent. Overexpression of the alpha1 subunit of T-type Ca2+ channels in human astrocytoma, neuroblastoma and renal tumor cell lines enhanced proliferation of these cells. In contrast, targeting of the alpha1 subunit of the T-type calcium channel via siRNA decreased proliferation of these cells. A Ca2+ oscillatory model is proposed involving potassium channels, Ca2+ stores and Ca2+ exchangers/transporters. A review of T-type channel blockers is presented, with a focus on mibefradil-induced inhibition of proliferation. The development of newer blockers with higher selectivity and less potential side effects are discussed. The conclusion reached is that calcium channel blockers serve as a potential therapeutic approach for tumors whose proliferation depends on T-type calcium channel expression.     

Y3+ block demonstrates an intracellular activation gate for the alpha1G T-type Ca2+ channel

Classical electrophysiology and contemporary crystallography suggest that the activation gate of voltage-dependent channels is on the intracellular side, but a more extracellular "pore gate" has also been proposed. We have used the voltage dependence of block by extracellular Y(3+) as a tool to locate the activation gate of the alpha1G (Ca(V)3.1) T-type calcium channel. Y(3+) block exhibited no clear voltage dependence from -40 to +40 mV (50% block at 25 nM), but block was relieved rapidly by stronger depolarization. Reblock of the open channel, reflected in accelerated tail currents, was fast and concentration dependent. Closed channels were also blocked by Y(3+) at a concentration-dependent rate, only eightfold slower than open-channel block. When extracellular Ca(2+) was replaced with Ba(2+), the rate of open block by Y(3+) was unaffected, but closed block was threefold faster than in Ca(2+), suggesting the slower closed-block rate reflects ion-ion interactions in the pore rather than an extracellularly located gate. Since an extracellular blocker can rapidly enter the closed pore, the primary activation gate must be on the intracellular side of the selectivity filter.     

Gating kinetics of the alpha1I T-type calcium channel.

The alpha1I T-type calcium channel inactivates almost 10-fold more slowly than the other family members (alpha1G and alpha1H) or most native T-channels. We have examined the underlying mechanisms using whole-cell recordings from rat alpha1I stably expressed in HEK293 cells. We found several kinetic differences between alpha1G and alpha1I, including some properties that at first appear qualitatively different. Notably, alpha1I tail currents require two or even three exponentials, whereas alpha1G tails were well described by a single exponential over a wide voltage range. Also, closed-state inactivation is more significant for alpha1I, even for relatively strong depolarizations. Despite these differences, gating of alpha1I can be described by the same kinetic scheme used for alpha1G, where voltage sensor movement is allosterically coupled to inactivation. Nearly all of the rate constants in the model are 5-12-fold slower for alpha1I, but the microscopic rate for channel closing is fourfold faster. This suggests that T-channels share a common gating mechanism, but with considerable quantitative variability.     

Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Cav3.2 T-type channel

Voltage-activated Ca²⁺ channels are membrane protein machinery performing selective permeation of external calcium ions. The main Ca²⁺ selective filters of all high-voltage-activated Ca²⁺ channel isoforms are commonly composed of four Glu residues (EEEE), while those of low-voltage-activated T-type Ca²⁺ channel isoforms are made up of two Glu and two Asp residues (EEDD). We here investigate how the Asp residues at the pore loops of domains III and IV affect biophysical properties of the Ca_v3.2 channel. Electrophysiological characterization of the pore mutant channels in which the pore Asp residue(s) were replaced with Glu, showed that both Asp residues critically control the biophysical properties of Ca_v3.2, including relative permeability between Ba²⁺ and Ca²⁺, anomalous mole fraction effect (AMFE), voltage dependency of channel activation, Cd²⁺ blocking sensitivity, and pH effects, in distinctive ways.