Single channel measurements demonstrate the voltage dependence of permeation through N-type and L-type CaV channels

The delivery of Ca²⁺ into cells by Ca_V channels provides the trigger for many cellular actions, such as cardiac muscle contraction and neurotransmitter release. Thus, a full understanding of Ca²⁺ permeation through these channels is critical. Using whole-cell voltage-clamp recordings, we recently demonstrated that voltage modulates the apparent affinity of N-type (Ca_V2.2) channels for permeating Ca²⁺ and Ba²⁺ ions. While we took many steps to ensure the high fidelity of our recordings, problems can occur when Ca_V currents become large and fast, or when currents run down. Thus, we use here single channel recordings to further test the hypothesis that permeating ions interact with N-type channels in a voltage-dependent manner. We also examined L-type (Ca_V1.2) channels to determine if these channels also exhibit voltage-dependent permeation. Like our whole-cell data, we find that voltage modulates N-channel affinity for Ba²⁺ at voltages > 0 mV, but has little or no effect at voltages < 0 mV. Furthermore, we demonstrate that permeation through L-channel is also modulated by voltage. Thus, voltage-dependence may be a common feature of divalent cation permeation through Ca_V1 and Ca_V2 channels (i.e. high-voltage activated Ca_V channels). The voltage dependence of Ca_V1 channel permeation is likely a mechanism mediating sustained Ca²⁺ influx during the plateau phase of the cardiac action potential.     

Single channel measurements demonstrate the voltage dependence of permeation through N-type and L-type CaV channels

The delivery of Ca²⁺ into cells by Ca_V channels provides the trigger for many cellular actions, such as cardiac muscle contraction and neurotransmitter release. Thus, a full understanding of Ca²⁺ permeation through these channels is critical. Using whole-cell voltage-clamp recordings, we recently demonstrated that voltage modulates the apparent affinity of N-type (Ca_V2.2) channels for permeating Ca²⁺ and Ba²⁺ ions. While we took many steps to ensure the high fidelity of our recordings, problems can occur when Ca_V currents become large and fast, or when currents run down. Thus, we use here single channel recordings to further test the hypothesis that permeating ions interact with N-type channels in a voltage-dependent manner. We also examined L-type (Ca_V1.2) channels to determine if these channels also exhibit voltage-dependent permeation. Like our whole-cell data, we find that voltage modulates N-channel affinity for Ba²⁺ at voltages > 0 mV, but has little or no effect at voltages < 0 mV. Furthermore, we demonstrate that permeation through L-channel is also modulated by voltage. Thus, voltage-dependence may be a common feature of divalent cation permeation through Ca_V1 and Ca_V2 channels (i.e. high-voltage activated Ca_V channels). The voltage dependence of Ca_V1 channel permeation is likely a mechanism mediating sustained Ca²⁺ influx during the plateau phase of the cardiac action potential.     

A Single Amino Acid Change in CaV1.2 Channels Eliminates the Permeation and Gating Differences Between Ca2+ and Ba2+

Glutamate scanning mutagenesis was used to assess the role of the calcicludine binding segment in regulating channel permeation and gating using both Ca²⁺ and Ba²⁺ as charge carriers. As expected, wild-type Ca_V1.2 channels had a Ba²⁺ conductance ~2× that in Ca²⁺ (G_Ba/G_Ca = 2) and activation was ~10 mV more positive in Ca²⁺ vs. Ba²⁺. Of the 11 mutants tested, F1126E was the only one that showed unique permeation and gating properties compared to the wild type. F1126E equalized the Ca_V1.2 channel conductance (G_Ba/G_Ca = 1) and activation voltage dependence between Ca²⁺ and Ba²⁺. Ba²⁺ permeation was reduced because the interactions among multiple Ba²⁺ ions and the pore were specifically altered for F1126E, which resulted in Ca²⁺-like ionic conductance and unitary current. However, the high-affinity block of monovalent cation flux was not altered for either Ca²⁺ or Ba²⁺. The half-activation voltage of F1126E in Ba²⁺ was depolarized to match that in Ca²⁺, which was unchanged from that in the wild type. As a result, the voltages for half-activation and half-inactivation of F1126E in Ba²⁺ and Ca²⁺ were similar to those of wild-type in Ca²⁺. This effect was specific to F1126E since F1126A did not affect the half-activation voltage in either Ca²⁺ or Ba²⁺. These results indicate that residues in the outer vestibule of the Ca_V1.2 channel pore are major determinants of channel gating, selectivity, and permeation.     

The HOOK region of β subunits controls gating of voltage-gated Ca2+ channels by electrostatically interacting with plasma membrane

Recently, we showed that the HOOK region of the β2 subunit electrostatically interacts with the plasma membrane and regulates the current inactivation and phosphatidylinositol 4,5-bisphosphate (PIP₂) sensitivity of voltage-gated Ca²⁺ (Ca_V) 2.2 channels. Here, we report that voltage-dependent gating and current density of the Ca_V2.2 channels are also regulated by the HOOK region of the β2 subunit. The HOOK region can be divided into 3 domains: S (polyserine), A (polyacidic), and B (polybasic). We found that the A domain shifted the voltage-dependent inactivation and activation of Ca_V2.2 channels to more hyperpolarized and depolarized voltages, respectively, whereas the B domain evoked these responses in the opposite directions. In addition, the A domain decreased the current density of the Ca_V2.2 channels, while the B domain increased it. Together, our data demonstrate that the flexible HOOK region of the β2 subunit plays an important role in determining the overall Ca_V channel gating properties.     

Voltage-dependent regulation of CaV2.2 channels by Gq-coupled receptor is facilitated by membrane-localized β subunit

G protein–coupled receptors (GPCRs) signal through molecular messengers, such as Gβγ, Ca²⁺, and phosphatidylinositol 4,5-bisphosphate (PIP₂), to modulate N-type voltage-gated Ca²⁺ (Ca_V2.2) channels, playing a crucial role in regulating synaptic transmission. However, the cellular pathways through which G_qPCRs inhibit Ca_V2.2 channel current are not completely understood. Here, we report that the location of Ca_V β subunits is key to determining the voltage dependence of Ca_V2.2 channel modulation by G_qPCRs. Application of the muscarinic agonist oxotremorine-M to tsA-201 cells expressing M₁ receptors, together with Ca_V N-type α1B, α2δ1, and membrane-localized β2a subunits, shifted the current-voltage relationship for Ca_V2.2 activation 5 mV to the right and slowed current activation. Muscarinic suppression of Ca_V2.2 activity was relieved by strong depolarizing prepulses. Moreover, when the C terminus of β-adrenergic receptor kinase (which binds Gβγ) was coexpressed with N-type channels, inhibition of Ca_V2.2 current after M₁ receptor activation was markedly reduced and delayed, whereas the delay between PIP₂ hydrolysis and inhibition of Ca_V2.2 current was decreased. When the Gβγ-insensitive Ca_V2.2 α1C-1B chimera was expressed, voltage-dependent inhibition of calcium current was virtually abolished, suggesting that M₁ receptors act through Gβγ to inhibit Ca_V2.2 channels bearing membrane-localized Ca_V β2a subunits. Expression of cytosolic β subunits such as β2b and β3, as well as the palmitoylation-negative mutant β2a(C3,4S), reduced the voltage dependence of M₁ muscarinic inhibition of Ca_V2.2 channels, whereas it increased inhibition mediated by PIP₂ depletion. Together, our results indicate that, with membrane-localized Ca_V β subunits, Ca_V2.2 channels are subject to Gβγ-mediated voltage-dependent inhibition, whereas cytosol-localized β subunits confer more effective PIP₂-mediated voltage-independent regulation. Thus, the voltage dependence of G_qPCR regulation of calcium channels can be determined by the location of isotype-specific Ca_V β subunits.     

Simulation of the effect of an external GHz electric field on the potential energy profile of Ca2+ ions in the selectivity filter of the CaVAb channel

Ca_V channels are transmembrane proteins that mediate and regulate ion fluxes across cell membranes, and they are activated in response to action potentials to allow Ca²⁺ influx. Since ion channels are composed of charge or polar groups, an external alternating electric field may affect the ion‐selective membrane transport and the performance of the channel. In this article, we have investigated the effect of an external GHz electric field on the dynamics of calcium ions in the selectivity filter of the Ca_VAb channel. Molecular dynamics (MD) simulations and the potential of mean force (PMF) calculations were carried out, via the umbrella sampling method, to determine the free energy profile of Ca²⁺ ions in the Ca_VAb channels in presence and absence of an external field. Exposing Ca_VAb channel to 1, 2, 3, 4, and 5 GHz electric fields increases the depth of the potential energy well and this may result in an increase in the affinity and strength of Ca²⁺ ions to binding sites in the selectivity filter the channel. This increase of strength of Ca²⁺ ions binding in the selectivity filter may interrupt the mechanism of Ca²⁺ ion conduction, and leads to a reduction of Ca²⁺ ion permeation through the Ca_VAb channel.     

Spermine biphasically affects N-type calcium channel currents in adult dorsal root ganglion neurons of the rat

Spermine (Spe) is a polyamine co-secreted with neurotransmitters. In this work its effects on N-type Ca²⁺ channel (Ca_V2.2) have been studied on adult sensory neurons of the rat by means of whole-cell patch-clamp. Spe exerted biphasic effects when added to the external solution: at 500 μM decreased N-type Ca²⁺ channel currents, reducing the maximum whole-cell conductance, shifting the activation curve to the right on the voltage axes and decreasing its slope; conversely, at lower concentration (500 nM) Spe induced completely opposite effects. In 62% of the neurons the inhibitory effects were accompanied by a slowing down of the activation kinetics relieved by a conditioning pre-pulse to + 50 mV. The biphasic effects and their rapid onset and offset time course may be explained if multiple sites of action with a different affinity for Spe are present directly on the channel. The effects of Spe on HVA Ca²⁺ currents were strongly dependent on [Ca²⁺]_ext, high [Ca²⁺] powerfully reducing Spe effects. This may be explained if we take into account that as Spe has four positive charges at physiological pH; it may compete with divalent cations for some negatively charged regulatory sites. In these experiments, Spe was effective at concentrations possibly reached in physiological conditions.     

Microscopic elements of electrical excitation in Chara: Transient activity of Cl− channels in the plasma membrane

The plasma membrane of Chara corallina was made accessible for patch pipettes by cutting a small window through the cell wall of plasmolyzed internodal cells. With pipettes containing Cl^– as Ca²⁺ or Ba²⁺ (50 or 100 mm), but not as Mg²⁺ or K⁺ salt, it was possible to record in the cell-attached mode for long periods with little channel activity, randomly interspersed with intervals of transient activation of two Cl^– channel types (cord conductance at +50 mV: 52 and 16 pS, respectively). During these periods of transient channel activity, variable numbers (up to some 10) of the two Cl^– channel types activated and again inactivated over several 100 msec in a coordinated fashion. Transient Cl channel activity was favored by voltages positive of the free running membrane voltage (> –45 mV); but positive voltage alone was neither a sufficient nor a necessary condition for activtion of these channels. Neither type of Cl^– channel was markedly voltage dependent. A third, nonselective 4 pS channel is a candidate for Ca²⁺ translocation. The activity of this channel does not correlate in time with the transient activity of the Cl^– channels. The entire set of results is consistent with the following microscopic mechanism of action potentials in Chara, concerning the role of Ca²⁺ and Cl^– for triggering and time course: Ca²⁺ uptake does not activate Cl^– channels directly but first supplies a membrane-associated population of Ca²⁺ storage sites. Depolarization enhances discharge of Ca²⁺ from these elements (none or few under the patch pipette) resulting in a local and transient increase of free Ca²⁺ concentration ([Ca²⁺]_cyt) at the inner side of the membrane before being scavenged by the cytoplasmic Ca²⁺ buffer system. In turn, the transient rise in [Ca²⁺]_cyt causes the transient activity of those Cl^– channels, which are more likely to open at an elevated Ca²⁺ concentration.The financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.     

Large Conductance Ca2+-Activated K+ Channels in Human Meningioma Cells

Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures. In whole cells, depolarization to voltages higher than +80 mV evoked a large K⁺ outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC₅₀= 5.3 mm). Raising the internal Ca²⁺ from 10 nm to 2 mm shifted the voltage of half-maximum activation (V _1/2) of the K⁺ current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca²⁺-activated (BK _Ca ) K⁺ channel with a conductance of 296 pS (130 mm K⁺ at both sides of the patch). V _1/2 of single-channel currents was +6, −12, −46, and −68 mV in the presence of 1, 10, 100, and 1000 μm Ca²⁺, respectively, at the internal face of the patch. In cell-attached patches the open probability (P _o ) of BK _Ca channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K⁺ currents with 10 nm Ca²⁺ in the pipette. Application of 20 μm cytochalasin D increased P _o of BK _Ca channels in cell-attached patches within minutes. These data suggest that the activation of BK _Ca channels in meningioma cells does not only depend on voltage and internal Ca²⁺ but is also controlled by the cytoskeleton. Received 18 June 1999/Revised: 18 January 2000     

Intracellular Ca(2+) oscillations induced by over-expressed Ca(V)3.1 T-type Ca(2+) channels in NG108-15 cells

T-type Ca²⁺ channel family includes three subunits Ca_V3.1, Ca_V3.2 and Ca_V3.3 and have been shown to control burst firing and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in neurons. Here, we investigated whether Ca_V3.1 channels could generate a pacemaker current and contribute to cell excitability. Ca_V3.1 clones were over-expressed in the neuronal cell line NG108-15. Ca_V3.1 channel expression induced repetitive action potentials, generating spontaneous membrane potential oscillations (MPOs) and concomitant [Ca²⁺]_i oscillations. These oscillations were inhibited by T-type channels antagonists and were present only if the membrane potential was around −61 mV. [Ca²⁺]_i oscillations were critically dependent on Ca²⁺ influx through Ca_V3.1 channels and did not involve Ca²⁺ release from the endoplasmic reticulum. The waveform and frequency of the MPOs are constrained by electrophysiological properties of the Ca_V3.1 channels. The trigger of the oscillations was the Ca_V3.1 window current. This current induced continuous [Ca²⁺]_i increase at −60 mV that depolarized the cells and triggered MPOs. Shifting the Ca_V3.1 window current potential range by increasing the external Ca²⁺ concentration resulted in a corresponding shift of the MPOs threshold. The hyperpolarization-activated cation current (I_h) was not required to induce MPOs, but when expressed together with Ca_V3.1 channels, it broadened the membrane potential range over which MPOs were observed. Overall, the data demonstrate that the Ca_V3.1 window current is critical in triggering intrinsic electrical and [Ca²⁺]_i oscillations.     

Arachidonic acid inhibition of L-type calcium (CaV1.3b) channels varies with accessory CaVβ subunits

Arachidonic acid (AA) inhibits the activity of several different voltage-gated Ca²⁺ channels by an unknown mechanism at an unknown site. The Ca²⁺ channel pore-forming subunit (Ca_Vα₁) is a candidate for the site of AA inhibition because T-type Ca²⁺ channels, which do not require accessory subunits for expression, are inhibited by AA. Here, we report the unanticipated role of accessory Ca_Vβ subunits on the inhibition of Ca_V1.3b L-type (L-) current by AA. Whole cell Ba²⁺ currents were measured from recombinant channels expressed in human embryonic kidney 293 cells at a test potential of −10 mV from a holding potential of −90 mV. A one-minute exposure to 10 µM AA inhibited currents with β_1b, β₃, or β₄ 58, 51, or 44%, respectively, but with β_2a only 31%. At a more depolarized holding potential of −60 mV, currents were inhibited to a lesser degree. These data are best explained by a simple model where AA stabilizes Ca_V1.3b in a deep closed-channel conformation, resulting in current inhibition. Consistent with this hypothesis, inhibition by AA occurred in the absence of test pulses, indicating that channels do not need to open to become inhibited. AA had no effect on the voltage dependence of holding potential–dependent inactivation or on recovery from inactivation regardless of Ca_Vβ subunit. Unexpectedly, kinetic analysis revealed evidence for two populations of L-channels that exhibit willing and reluctant gating previously described for Ca_V2 channels. AA preferentially inhibited reluctant gating channels, revealing the accelerated kinetics of willing channels. Additionally, we discovered that the palmitoyl groups of β_2a interfere with inhibition by AA. Our novel findings that the Ca_Vβ subunit alters kinetic changes and magnitude of inhibition by AA suggest that Ca_Vβ expression may regulate how AA modulates Ca²⁺-dependent processes that rely on L-channels, such as gene expression, enzyme activation, secretion, and membrane excitability.     

Divergent Ca2+/calmodulin feedback regulation of CaV1 and CaV2 voltage-gated calcium channels evolved in the common ancestor of Placozoa and Bilateria

Ca_V1 and Ca_V2 voltage-gated calcium channels evolved from an ancestral Ca_V1/2 channel via gene duplication somewhere near the stem animal lineage. The divergence of these channel types led to distinguishing functional properties that are conserved among vertebrates and bilaterian invertebrates and contribute to their unique cellular roles. One key difference pertains to their regulation by calmodulin (CaM), wherein bilaterian Ca_V1 channels are uniquely subject to pronounced, buffer-resistant Ca²⁺/CaM-dependent inactivation, permitting negative feedback regulation of calcium influx in response to local cytoplasmic Ca²⁺ rises. Early diverging, nonbilaterian invertebrates also possess Ca_V1 and Ca_V2 channels, but it is unclear whether they share these conserved functional features. The most divergent animals to possess both Ca_V1 and Ca_V2 channels are placozoans such as Trichoplax adhaerens, which separated from other animals over 600 million years ago shortly after their emergence. Hence, placozoans can provide important insights into the early evolution of Ca_V1 and Ca_V2 channels. Here, we build upon previous characterization of Trichoplax Ca_V channels by determining the cellular expression and ion-conducting properties of the Ca_V1 channel orthologue, TCa_V1. We show that TCa_V1 is expressed in neuroendocrine-like gland cells and contractile dorsal epithelial cells. In vitro, this channel conducts dihydropyridine-insensitive, high-voltage–activated Ca²⁺ currents with kinetics resembling those of rat Ca_V1.2 but with left-shifted voltage sensitivity for activation and inactivation. Interestingly, TCa_V1, but not TCa_V2, exhibits buffer-resistant Ca²⁺/CaM-dependent inactivation, indicating that this functional divergence evolved prior to the emergence of bilaterian animals and may have contributed to their unique adaptation for cytoplasmic Ca²⁺ signaling within various cellular contexts.     

Phospholemman Modulates the Gating of Cardiac L-Type Calcium Channels

Ca²⁺ entry through L-type calcium channels (Ca_V1.2) is critical in shaping the cardiac action potential and initiating cardiac contraction. Modulation of Ca_V1.2 channel gating directly affects myocyte excitability and cardiac function. We have found that phospholemman (PLM), a member of the FXYD family and regulator of cardiac ion transport, coimmunoprecipitates with Ca_V1.2 channels from guinea pig myocytes, which suggests PLM is an endogenous modulator. Cotransfection of PLM in HEK293 cells slowed Ca_V1.2 current activation at voltages near the threshold for activation, slowed deactivation after long and strong depolarizing steps, enhanced the rate and magnitude of voltage-dependent inactivation (VDI), and slowed recovery from inactivation. However, Ca²⁺-dependent inactivation was not affected. Consistent with slower channel closing, PLM significantly increased Ca²⁺ influx via Ca_V1.2 channels during the repolarization phase of a human cardiac action potential waveform. Our results support PLM as an endogenous regulator of Ca_V1.2 channel gating. The enhanced VDI induced by PLM may help protect the heart under conditions such as ischemia or tachycardia where the channels are depolarized for prolonged periods of time and could induce Ca²⁺ overload. The time and voltage-dependent slowed deactivation could represent a gating shift that helps maintain Ca²⁺ influx during the cardiac action potential waveform plateau phase.     

Grina/TMBIM3 modulates voltage-gated CaV2.2 Ca2+ channels in a G-protein-like manner

Grina/TMBIM3 is a poorly characterized transmembrane protein with a broad expression pattern in mammals and with a very ancient origin within eukaryotes. Although initially characterized as an NMDA-receptor associated subunit, there is increasing evidence that Grina/TMBIM3 is involved in the unfolded protein response and controls apoptosis via regulation of Ca²⁺ homeostasis. Here, we investigate a putative direct interaction of Grina/TMBIM3 with voltage gated Ca²⁺ channels, in particular with the Ca_V2.2 α1-subunit and describe its modulatory effects on the current through Ca_V2.2 N-type channels. Direct interaction was confirmed by co-immunoprecipitation studies and membrane localization was proven. Co-expression of Grina/TMBIM3 with Ca_V2.2 channels resulted in a significant decrease of the current amplitude and in a slowing of the kinetics of current activation. This effect was accompanied by a significant shift of the voltage dependencies of activation time constants towards more depolarized voltages. Application of a stimulus protocol including a strong depolarizing pulse relieved inhibition of current amplitude by Grina/TMBIM3. When Grina/TMBIM3 was present, inactivation by an action potential-like train of pulses was diminished. Both observations resemble mechanisms that are well-studied modulatory effects of G-protein βγ subunits on Ca_V2 channels. The impact of Grina/TMBIM3 and G-protein βγ subunits are rather comparable with respect to suppression of current amplitude and slowing of activation kinetics. Furthermore, both modulators had the same effect on current inactivation when evoked by an action potential-like train of pulses.     

Electrical coupling between the human serotonin transporter and voltage-gated Ca channels

Monoamine transporters have been implicated in dopamine or serotonin release in response to abused drugs such as methamphetamine or ecstasy (MDMA). In addition, monoamine transporters show substrate-induced inward currents that may modulate excitability and Ca²⁺ mobilization, which could also contribute to neurotransmitter release. How monoamine transporters modulate Ca²⁺ permeability is currently unknown. We investigate the functional interaction between the human serotonin transporter (hSERT) and voltage-gated Ca²⁺ channels (Ca_V). We introduce an excitable expression system consisting of cultured muscle cells genetically engineered to express hSERT. Both 5HT and S(+)MDMA depolarize these cells and activate the excitation-contraction (EC)-coupling mechanism. However, hSERT substrates fail to activate EC-coupling in Ca_V1.1-null muscle cells, thus implicating Ca²⁺ channels. Ca_V1.3 and Ca_V2.2 channels are natively expressed in neurons. When these channels are co-expressed with hSERT in HEK293T cells, only cells expressing the lower-threshold L-type Ca_V1.3 channel show Ca²⁺ transients evoked by 5HT or S(+)MDMA. In addition, the electrical coupling between hSERT and Ca_V1.3 takes place at physiological 5HT concentrations. The electrical coupling between monoamine neurotransmitter transporters and Ca²⁺ channels such as Ca_V1.3 is a novel mechanism by which endogenous substrates (neurotransmitters) or exogenous substrates (like ecstasy) could modulate Ca²⁺-driven signals in excitable cells.     

Role of the beta1 subunit in large-conductance Ca(2+)-activated K(+) channel gating energetics. Mechanisms of enhanced Ca(2+) sensitivity

Over the past few years, it has become clear that an important mechanism by which large-conductance Ca²⁺-activated K⁺ channel (BK_Ca) activity is regulated is the tissue-specific expression of auxiliary β subunits. The first of these to be identified, β1, is expressed predominately in smooth muscle and causes dramatic effects, increasing the apparent affinity of the channel for Ca²⁺ 10-fold at 0 mV, and shifting the range of voltages over which the channel activates −80 mV at 9.1 μM Ca²⁺. With this study, we address the question: which aspects of BK_Ca gating are altered by β1 to bring about these effects: Ca²⁺ binding, voltage sensing, or the intrinsic energetics of channel opening? The approach we have taken is to express the β1 subunit together with the BK_Ca α subunit in Xenopus oocytes, and then to compare β1''s steady state effects over a wide range of Ca²⁺ concentrations and membrane voltages to those predicted by allosteric models whose parameters have been altered to mimic changes in the aspects of gating listed above. The results of our analysis suggest that much of β1''s steady state effects can be accounted for by a reduction in the intrinsic energy the channel must overcome to open and a decrease in its voltage sensitivity, with little change in the affinity of the channel for Ca²⁺ when it is either open or closed. Interestingly, however, the small changes in Ca²⁺ binding affinity suggested by our analysis (K_c 7.4 μM → 9.6 μM; K_o = 0.80 μM → 0.65 μM) do appear to be functionally important. We also show that β1 affects the mSlo conductance–voltage relation in the essential absence of Ca²⁺, shifting it +20 mV and reducing its apparent gating charge 38%, and we develop methods for distinguishing between alterations in Ca²⁺ binding and other aspects of BK_Ca channel gating that may be of general use.     

Selective pressure modulation of synaptic voltage‐dependent calcium channels—involvement in HPNS mechanism

Exposure to hyperbaric pressure (HP) exceeding 100 msw (1.1 MPa) is known to cause a constellation of motor and cognitive impairments named high‐pressure neurological syndrome (HPNS), considered to be the result of synaptic transmission alteration. Long periods of repetitive HP exposure could be an occupational risk for professional deep‐sea divers. Previous studies have indicated the modulation of presynaptic Ca²⁺ currents based on synaptic activity modified by HP. We have recently demonstrated that currents in genetically identified cellular voltage‐dependent Ca²⁺ channels (VDCCs), Ca_V1.2 and Ca_V3.2 are selectively affected by HP. This work further elucidates the HPNS mechanism by examining HP effect on Ca²⁺ currents in neuronal VDCCs, Ca_V2.2 and Ca_V2.1, which are prevalent in presynaptic terminals, expressed in Xenopus oocytes. HP augmented the Ca_V2.2 current amplitude, much less so in a channel variation containing an additional modulatory subunit, and had almost no effect on the Ca_V2.1 currents. HP differentially affected the channels' kinetics. It is, therefore, suggested that HPNS signs and symptoms arise, at least in part, from pressure modulation of various VDCCs.     

Mechanisms of a Human Skeletal Myotonia Produced by Mutation in the C-Terminus of NaV1.4: Is Ca2+ Regulation Defective?

Mutations in the cytoplasmic tail (CT) of voltage gated sodium channels cause a spectrum of inherited diseases of cellular excitability, yet to date only one mutation in the CT of the human skeletal muscle voltage gated sodium channel (hNa_V1.4_F1705I) has been linked to cold aggravated myotonia. The functional effects of altered regulation of hNa_V1.4_F1705I are incompletely understood. The location of the hNa_V1.4_F1705I in the CT prompted us to examine the role of Ca²⁺ and calmodulin (CaM) regulation in the manifestations of myotonia. To study Na channel related mechanisms of myotonia we exploited the differences in rat and human Na_V1.4 channel regulation by Ca²⁺ and CaM. hNa_V1.4_F1705I inactivation gating is Ca²⁺-sensitive compared to wild type hNa_V1.4 which is Ca²⁺ insensitive and the mutant channel exhibits a depolarizing shift of the V_1/2 of inactivation with CaM over expression. In contrast the same mutation in the rNa_V1.4 channel background (rNa_V1.4_F1698I) eliminates Ca²⁺ sensitivity of gating without affecting the CaM over expression induced hyperpolarizing shift in steady-state inactivation. The differences in the Ca²⁺ sensitivity of gating between wild type and mutant human and rat Na_V1.4 channels are in part mediated by a divergence in the amino acid sequence in the EF hand like (EFL) region of the CT. Thus the composition of the EFL region contributes to the species differences in Ca²⁺/CaM regulation of the mutant channels that produce myotonia. The myotonia mutation F1705I slows I_Na decay in a Ca²⁺-sensitive fashion. The combination of the altered voltage dependence and kinetics of I_Na decay contribute to the myotonic phenotype and may involve the Ca²⁺-sensing apparatus in the CT of Na_V1.4.     

Apamin Boosting of Synaptic Potentials in CaV2.3 R-Type Ca2+ Channel Null Mice

SK2- and K_V4.2-containing K⁺ channels modulate evoked synaptic potentials in CA1 pyramidal neurons. Each is coupled to a distinct Ca²⁺ source that provides Ca²⁺-dependent feedback regulation to limit AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated postsynaptic depolarization. SK2-containing channels are activated by Ca²⁺ entry through NMDARs, whereas K_V4.2-containing channel availability is increased by Ca²⁺ entry through SNX-482 (SNX) sensitive Ca_V2.3 R-type Ca²⁺ channels. Recent studies have challenged the functional coupling between NMDARs and SK2-containing channels, suggesting that synaptic SK2-containing channels are instead activated by Ca²⁺ entry through R-type Ca²⁺ channels. Furthermore, SNX has been implicated to have off target affects, which would challenge the proposed coupling between R-type Ca²⁺ channels and K_V4.2-containing K⁺ channels. To reconcile these conflicting results, we evaluated the effect of SK channel blocker apamin and R-type Ca²⁺ channel blocker SNX on evoked excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal neurons from Ca_V2.3 null mice. The results show that in the absence of Ca_V2.3 channels, apamin application still boosted EPSPs. The boosting effect of Ca_V2.3 channel blockers on EPSPs observed in neurons from wild type mice was not observed in neurons from Ca_V2.3 null mice. These data are consistent with a model in which SK2-containing channels are functionally coupled to NMDARs and K_V4.2-containing channels to Ca_V2.3 channels to provide negative feedback regulation of EPSPs in the spines of CA1 pyramidal neurons.     

G protein inhibition of CaV2 calcium channels

Voltage-gated Ca²⁺ channels translate the electrical inputs of excitable cells into biochemical outputs by controlling influx of the ubiquitous second messenger Ca²⁺. As such the channels play pivotal roles in many cellular functions including the triggering of neurotransmitter and hormone release by Ca_V2.1 (P/Q-type) and Ca_V2.2 (N-type) channels. It is well established that G protein coupled receptors (GPCRs) orchestrate precise regulation neurotransmitter and hormone release through inhibition of Ca_V2 channels. Although the GPCRs recruit a number of different pathways, perhaps the most prominent, and certainly most studied among these is the so-called voltage-dependent inhibition mediated by direct binding of Gβγ to the α₁ subunit of Ca_V2 channels. This article will review the basics of Ca²⁺-channels and G protein signaling, and the functional impact of this now classical inhibitory mechanism on channel function. It will also provide an update on more recent developments in the field, both related to functional effects and crosstalk with other signaling pathways, and advances made toward understanding the molecular interactions that underlie binding of Gβγ to the channel and the voltage-dependence that is a signature characteristic of this mechanism.