L-type channel inactivation balances the increased peak calcium current due to absence of Rad in cardiomyocytes

The L-type Ca²⁺ channel (LTCC) provides trigger calcium to initiate cardiac contraction in a graded fashion that is regulated by L-type calcium current (I_Ca,L) amplitude and kinetics. Inactivation of LTCC is controlled to fine-tune calcium flux and is governed by voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). Rad is a monomeric G protein that regulates I_Ca,L and has recently been shown to be critical to β-adrenergic receptor (β-AR) modulation of I_Ca,L. Our previous work showed that cardiomyocyte-specific Rad knockout (cRadKO) resulted in elevated systolic function, underpinned by an increase in peak I_Ca,L, but without pathological remodeling. Here, we sought to test whether Rad-depleted LTCC contributes to the fight-or-flight response independently of β-AR function, resulting in I_Ca,L kinetic modifications to homeostatically balance cardiomyocyte function. We recorded whole-cell I_Ca,L from ventricular cardiomyocytes from inducible cRadKO and control (CTRL) mice. The kinetics of I_Ca,L stimulated with isoproterenol in CTRL cardiomyocytes were indistinguishable from those of unstimulated cRadKO cardiomyocytes. CDI and VDI are both enhanced in cRadKO cardiomyocytes without differences in action potential duration or QT interval. To confirm that Rad loss modulates LTCC independently of β-AR stimulation, we crossed a β₁,β₂-AR double-knockout mouse with cRadKO, resulting in a Rad-inducible triple-knockout mouse. Deletion of Rad in cardiomyocytes that do not express β₁,β₂-AR still yielded modulated I_Ca,L and elevated basal heart function. Thus, in the absence of Rad, increased Ca²⁺ influx is homeostatically balanced by accelerated CDI and VDI. Our results indicate that the absence of Rad can modulate the LTCC without contribution of β₁,β₂-AR signaling and that Rad deletion supersedes β-AR signaling to the LTCC to enhance in vivo heart function.     

Voltage- and calcium-dependent inactivation in high voltage-gated Ca(2+) channels

Calcium influx into cardiac myocytes via voltage-gated Ca channels is a key step in initiating the contractile response. During prolonged depolarizations, toxic Ca(2+) overload is prevented by channel inactivation occurring through two different processes identified by their primary trigger: voltage or intracellular Ca(2+). In physiological situations, cardiac L-type (Ca(V)1.2) Ca(2+) channels inactivate primarily via Ca(2+)-dependent inactivation (CDI), while neuronal P/Q (Ca(V)2.1) Ca(2+) channels use preferentially voltage-dependent inactivation (VDI). In certain situations however, these two types of channels have been shown to be able to inactivate by both processes. From a structural view point, the rearrangement occurring during CDI and VDI is not precisely known, but functional studies have underlined the role played by at least 2 channel sequences: a C-terminal binding site for the Ca(2+) sensor calmodulin, essential for CDI, and the loop connecting domains I and II, essential for VDI. The conserved regulation of VDI and CDI by the auxiliary channel beta subunit strongly suggests that these two mechanisms may use a set of common protein-protein interactions that are influenced by the auxiliary subunit. We will review our current knowledge of these interactions. New data are presented on L-P/Q (Ca(V)1.2/Ca(V)2.1) channel chimera that confirm the role of the I-II loop in VDI and CDI, and reveal some of the essential steps in Ca(2+) channel inactivation.     

Rem-GTPase regulates cardiac myocyte L-type calcium current

Rationale: The L-type calcium channels (LTCC) are critical for maintaining Ca²⁺-homeostasis. In heterologous expression studies, the RGK-class of Ras-related G-proteins regulates LTCC function; however, the physiological relevance of RGK–LTCC interactions is untested.

Objective: In this report we test the hypothesis that the RGK protein, Rem, modulates native Ca²⁺ current (I_Ca,L) via LTCC in murine cardiomyocytes.

Methods and Results: Rem knockout mice (Rem^?/?) were engineered, and I_Ca,L and Ca²⁺-handling properties were assessed. Rem^?/? ventricular cardiomyocytes displayed increased I_Ca,L density. I_Ca,L activation was shifted positive on the voltage axis, and β-adrenergic stimulation normalized this shift compared with wild-type I_Ca,L. Current kinetics, steady-state inactivation, and facilitation was unaffected by Rem^?/?. Cell shortening was not significantly different. Increased I_Ca,L density in the absence of frank phenotypic differences motivated us to explore putative compensatory mechanisms. Despite the larger I_Ca,L density, Rem^?/? cardiomyocyte Ca²⁺ twitch transient amplitude was significantly less than that compared with wild type. Computer simulations and immunoblot analysis suggests that relative dephosphorylation of Rem^?/? LTCC can account for the paradoxical decrease of Ca²⁺ transients.

Conclusions: This is the first demonstration that loss of an RGK protein influences I_Ca,L in vivo in cardiac myocytes.     

Physiological modulation of voltage-dependent inactivation in the cardiac muscle L-type calcium channel: a modelling study

The inactivation of the L-type Ca²⁺ current is composed of voltage-dependent and calcium-dependent mechanisms. The relative contribution of these processes is still under dispute and the idea that the voltage-dependent inactivation could be subject to further modulation by other physiological processes had been ignored. This study sought to model physiological modulation of inactivation of the current in cardiac ventricular myocytes, based upon the recent detailed experimental data that separated total and voltage-dependent inactivation (VDI) by replacing extracellular Ca²⁺ with Mg²⁺ and monitoring L-type Ca²⁺ channel behaviour by outward K⁺ current flowing through the channel in the absence of inward current flow. Calcium-dependent inactivation (CDI) was based upon Ca²⁺ influx and formulated from data that was recorded during β-adrenergic stimulation of the myocytes. Ca²⁺ influx and its competition with non-selective monovalent cation permeation were also incorporated into channel permeation in the model. The constructed model could closely reproduce the experimental Ba²⁺ and Ca²⁺ current results under basal condition where no β-stimulation was added after a slight reduction of the development of fast voltage-dependent inactivation with depolarization. The model also predicted that under β-adrenergic stimulation voltage-dependent inactivation is lost and calcium-dependent inactivation largely compensates it. The developed model thus will be useful to estimate the respective roles of VDI and CDI of L-type Ca²⁺ channels in various physiological and pathological conditions of the heart which would otherwise be difficult to show experimentally.     

Separation of ionic currents in the somatic membrane of frog sensory neurons

Summary Electrical properties of isolated frog primary afferent neurons were examined by suction pipette technique, which combines internal perfusion with current or voltage clamp using a switching circuit with a single electrode. When K⁺ in the external and internal solutions was totally replaced with Cs⁺, extremely prolonged Ca spikes, lasting for 5 to 10 sec, and Na spikes, having a short plateau phase of 10 to 15 msec, were observed in Na⁺-free and Ca²⁺-free solutions, respectively. Under voltage clamp, Ca²⁺ current (I _Ca) appeared at around –30 mV and maximum peak current was elicited at about 0 mV. With increasing test pulses to the positive side,I _Ca became smaller and flattened but did not reverse. Increases of [Ca] _o induced a hyperbolic increase ofI _Ca and also shifted itsI-V curve along the voltage axis to the more positive direction. Internal perfusion of F^– blockedI _Ca time-dependently. The Ca channel was permeable to foreign divalent cations in the sequence ofI _Ca>I _Ba>I _SrI _Mn>I _Zn. Organic Ca-blockers equally depressed the divalent cation currents dose- and time-dependently without shifting theI-V relationships, while inorganic blockers suppressed these currents dose-dependently and the inhibition appeared much stronger in the order ofI _Ba=I _Sr>I _Ca>I _Mn=I _Zn.     

Inactivation of calcium channels in vascular smooth muscle myocytes

Many of the structural domains involved in Ca²⁺ channel (CACN) inactivation are also involved in determining their sensitivity to antagonist inhibition. We hypothesize that differences in inactivation properties and their structural determinants may suggest candidate domains as targets for the development of novel, selective antagonists. The characteristics of Ca²⁺ current (I_Ca) inactivation, steady-state inactivation (SSIN), and recovery from inactivation were studied in freshly dispersed smooth muscle cells from rabbit portal vein (RPV) using whole-cell, voltage-clamp methods. The time course of inactivation could be represented by two time constants. Increasing I_Ca by increasing [Ca²⁺]_o or with more negative holding potentials decreased both time constants. With Sr²⁺, Ba²⁺, or Na⁺ as the charge carrier, I_Ca inactivation was also represented by two time constants, both of which were larger than those found with Ca²⁺. With Ca²⁺, Sr²⁺, or Ba²⁺ as the charge carrier, both time constants had minimum values near the voltage associated with maximum current. When Na⁺ (140 mM) was the charge carrier, voltages for I_max (−20 mV) or τ_min (o mV) did not correspond. SSIN of I_Ca had a half-maximum voltage of −32±4 mV for Ca²⁺, −43 mV±5 mV for Sr²⁺, −41±5 mV for Ba²⁺, and −68±6 mV for Na⁺. The slope factor for SSIN per e-fold voltage change was 6.5±0.2 mV for Ca²⁺, 6.8±0.3 for Sr²⁺, and 6.6±0.2 for Ba²⁺, representing four equivalent charges. When Na⁺ or Li⁺ was the charge carrier, the slope factor was 13.5±0.7 mV, representing two equivalent charges. For I_Ca in rat left ventricular (rLV) myocytes, there was no difference in the slope factor of SSIN for Ca²⁺ and Na⁺. The rate of recovery of I_Ca from inactivation varied inversely with recovery voltage and was independent of the charge carrier. These results suggest that inactivation of I_Ca in PV myocytes possess an intrinsic voltage dependence that is modified by Ca²⁺. For RPV but not rLV I_Ca, the charge of the permeating ion confers the voltage-dependency of SSIN.     

Membrane Currents Carried by Ca, Sr, and Ba in Barnacle Muscle Fiber During Voltage Clamp

Membrane currents associated with voltage clamp of the giant muscle fibers of a barnacle, Balanus nubilus, were analyzed in terms of currents of the Ca and K channels. Although the activation of the K channel occurs more slowly than that of the Ca channel, both currents show a significant temporal overlap. The currents carried by Ca⁺⁺, Sr⁺⁺, or Ba⁺⁺ through the Ca channel were compared under the conditions at which this overlap was the least. When only one divalent cation is present in the solution, Ba⁺⁺ carries more current than Ca⁺⁺ or Sr⁺⁺ and the sequence of the current is Ba > Sr ≈ Ca. When the external solution contains a relatively high concentration of Co⁺⁺, which is a blocking agent for the Ca channel, inversion of the sequence occurs, to Ca > Sr > Ba. This is due to the fact that the blocking effect differs depending on which ion carries current through the Ca channel. The Ba current is most sensitive and the Ca current is least affected. Ba suppresses the current of the K channel, independently of its current-carrying function through the Ca channel.     

Molecular endpoints of Ca2+/calmodulin- and voltage-dependent inactivation of Cav1.3 channels

Ca²⁺/calmodulin- and voltage-dependent inactivation (CDI and VDI) comprise vital prototypes of Ca²⁺ channel modulation, rich with biological consequences. Although the events initiating CDI and VDI are known, their downstream mechanisms have eluded consensus. Competing proposals include hinged-lid occlusion of channels, selectivity filter collapse, and allosteric inhibition of the activation gate. Here, novel theory predicts that perturbations of channel activation should alter inactivation in distinctive ways, depending on which hypothesis holds true. Thus, we systematically mutate the activation gate, formed by all S6 segments within Ca_V1.3. These channels feature robust baseline CDI, and the resulting mutant library exhibits significant diversity of activation, CDI, and VDI. For CDI, a clear and previously unreported pattern emerges: activation-enhancing mutations proportionately weaken inactivation. This outcome substantiates an allosteric CDI mechanism. For VDI, the data implicate a “hinged lid–shield” mechanism, similar to a hinged-lid process, with a previously unrecognized feature. Namely, we detect a “shield” in Ca_V1.3 channels that is specialized to repel lid closure. These findings reveal long-sought downstream mechanisms of inactivation and may furnish a framework for the understanding of Ca²⁺ channelopathies involving S6 mutations.     

Expression of calcium channels in Xenopus oocyte injected with crab skeletal muscle fibre mRNA

Summary— Using the whole cell voltage-clamp technique and a Cl free and Na free Ba methane sulfonate solution, stage V and VI Xenopus oocytes demonstrated a Ba current (endogenous component) with a peak amplitude average of 6 nA (6 ± 2 nA). When oocytes were injected with crustacean skeletal muscle mRNA, an additional component of I_Ba could be detected (exogenous I_Ba). The latter current could be distinguished from the native one by several electrophysiological means: a peak amplitude average of 90 nA (90 ± 4 nA), activation potential threshold, steady state inactivation properties and sensitivity to Ca blockers. As shown by Jdaïâa and Guilbault in crustacean skeletal muscle fibres, exogenous I_Ba could be divided into two components: a “fast component” and a “slow component” probably passing through two types of Ca channels (fast and slow) since the peak Ba current voltage relationship was biphasic and the fast component of exogenous I_Ba was less sensitive than the slow to nifedipine. The features of the newly synthesized channels incorporated in the Xenopus oocyte membrane suggest that they may be associated with fast and slow channels, previously described in many preparations, particularly in crustacean skeletal muscle fibres.     

Expression of calcium channels in Xenopus oocyte injected with crab skeletal muscle fibre mRNA

Using the whole cell voltage-clamp technique and a Cl free and Na free Ba methane sulfonate solution, stage V and VI Xenopus oocytes demonstrated a Ba current (endogenous component) with a peak amplitude average of 6 nA (6 ± 2 nA). When oocytes were injected with crustacean skeletal muscle mRNA, an additional component of I_Ba could be detected (exogenous I_Ba). The latter current could be distinguished from the native one by several electrophysiological means: a peak amplitude average of 90 nA (90 ± 4 nA), activation potential threshold, steady state inactivation properties and sensitivity to Ca blockers. As shown by Jdaïâa and Guilbault in crustacean skeletal muscle fibres, exogenous I_Ba could be divided into two components: a “fast component” and a “slow component” probably passing through two types of Ca channels (fast and slow) since the peak Ba current voltage relationship was biphasic and the fast component of exogenous I_Ba was less sensitive than the slow to nifedipine. The features of the newly synthesized channels incorporated in the Xenopus oocyte membrane suggest that they may be associated with fast and slow channels, previously described in many preparations, particularly in crustacean skeletal muscle fibres.     

Dual effects of K ions upon the inactivation of the anomalous rectifier of the tunicate egg cell membrane

Summary Inactivation of the K inward current through the anomalous rectifier channel of the egg cell membrane of a tunicate,Halocynthia roretzi Drashe, was studied under voltage-clamp. The noise spectrum of the steady-state current recorded at hyperpolarized potentials was measured in solutions in which Na, Cs, Hydrazine, or Sr caused inactivation of the current. The unitary conductance estimated was independent of which cation caused inactivation. From the relation between the concentration of cations which caused inactivation and the extent of inactivation at fixed potentials, the binding of one inactivator to a channel was found to cause inactivation, and the potency of inactivation was Cs⁺>Hydrazine⁺>Na⁺>Li⁺, and Ba²⁺>Sr²⁺. The inactivation caused by Na⁺ was increased by K⁺ when [K] _o was lower than 20mm, but was decreased by K⁺ in higher K-ASW (artificial sea water). One K⁺ was found to inactivate the channel cooperatively with one Na⁺. Increase of inactivation by K⁺ was a dominant effect in Cs-ASW. The inactivation was explained quantitatively by a model assuming cooperative plugging by a monovalent inactivator and a K⁺.     

Systematic mapping of the state dependence of voltage- and Ca2+-dependent inactivation using simple open-channel measurements

The state from which channel inactivation occurs is both biologically and mechanistically critical. For example, preferential closed-state inactivation is potentiated in certain Ca²⁺ channel splice variants, yielding an enhancement of inactivation during action potential trains, which has important consequences for short-term synaptic plasticity. Mechanistically, the structural substrates of inactivation are now being resolved, yielding a growing library of molecular snapshots, ripe for functional interpretation. For these reasons, there is an increasing need for experimentally direct and systematic means of determining the states from which inactivation proceeds. Although many approaches have been devised, most rely upon numerical models that require detailed knowledge of channel-state topology and gating parameters. Moreover, prior strategies have only addressed voltage-dependent forms of inactivation (VDI), and have not been readily applicable to Ca²⁺-dependent inactivation (CDI), a vital form of regulation in numerous contexts. Here, we devise a simple yet systematic approach, applicable to both VDI and CDI, for semiquantitative mapping of the states from which inactivation occurs, based only on open-channel measurements. The method is relatively insensitive to the specifics of channel gating and does not require detailed knowledge of state topology or gating parameters. Rather than numerical models, we derive analytic equations that permit determination of the states from which inactivation occurs, based on direct manipulation of data. We apply this methodology to both VDI and CDI of Ca_V1.3 Ca²⁺ channels. VDI is found to proceed almost exclusively from the open state. CDI proceeds equally from the open and nearby closed states, but is disfavored from deep closed states distant from the open conformation. In all, these outcomes substantiate and enrich conclusions of our companion paper in this issue (Tadross et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910308) that deduces endpoint mechanisms of VDI and CDI in Ca_V1.3. More broadly, the methods introduced herein can be readily generalized for the analysis of other channel types.     

Interaction between calcium ions and surface charge as it relates to calcium currents

Summary Calcium ions affect the gating of Ca currents. Surface charge is involved but to what extent is unknown. We have examined this, using isolated nerve cell bodies ofHelix aspersa and the combined microelectrode-suction pipette method for voltage-clamp and internal perfusion. We found that Ba and Sr currents produced by substitution of these ions for extracellular Ca ions are activated at less positive potentials than Ca currents. Mg ions do not permeate the Ca channel and changes in [Mg]₀ produce shifts in the activation-potential curves that are comparable to the effects of changes in [Ba]₀ or [Sr]₀. Inactivation of Ba currents also occurs at less positive potentials. Perfusion intracellularly with EGTA reduced inactivation of Ca currents as a function of potential, but did not shift the inactivation-potential curve. Hence, Ca current-dependent inactivation which is blocked by intracellular EGTA probably does not involve a similar change of intracellular surface potential. The voltage shifts of activation and inactivation produced by extracellular divalent cations used singly or in mixtures can be described by the Gouy-Chapman theory for the diffuse double layer with binding (Gilbert & Ehrenstein, 1969; McLaughlin, Szabo & Eisenman, 1971). From the surface potential values and the Boltzman distribution, we have computed surface concentrations that predict the following experimental observations: 1) saturation of current-concentration relationships when surface potential is changing maximally; 2) the increase in peak current when Ca ions are replaced by Sr or Ba ions; and 3) the greater inhibitory effect of Mg onI _Ba thanI _Ca. Theory indicates that surface charge cannot be screened completely even at 1m [Mg]₀ and thus that Ca channel properties must be evaluated in the light of surface charge effects. For example, after correction for surface charge effects the relative permeabilities of Ca, Ba and Sr ions are equivalent. In the presence of Co ions, however, Ca ions are more permeable than Ba ions suggesting a channel binding site may be involved.     

Ion-dependent Inactivation of Barium Current through L-type Calcium Channels

It is widely believed that Ba²⁺ currents carried through L-type Ca²⁺ channels inactivate by a voltage- dependent mechanism similar to that described for other voltage-dependent channels. Studying ionic and gating currents of rabbit cardiac Ca²⁺ channels expressed in different subunit combinations in tsA201 cells, we found a phase of Ba²⁺ current decay with characteristics of ion-dependent inactivation. Upon a long duration (20 s) depolarizing pulse, I_Ba decayed as the sum of two exponentials. The slow phase (τ ≈ 6 s, 21°C) was parallel to a reduction of gating charge mobile at positive voltages, which was determined in the same cells. The fast phase of current decay (τ ≈ 600 ms), involving about 50% of total decay, was not accompanied by decrease of gating currents. Its amplitude depended on voltage with a characteristic U-shape, reflecting reduction of inactivation at positive voltages. When Na⁺ was used as the charge carrier, decay of ionic current followed a single exponential, of rate similar to that of the slow decay of Ba²⁺ current. The reduction of Ba²⁺ current during a depolarizing pulse was not due to changes in the concentration gradients driving ion movement, because Ba²⁺ entry during the pulse did not change the reversal potential for Ba²⁺. A simple model of Ca²⁺-dependent inactivation (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1993. J. Gen. Physiol. 102:1005–1030) robustly accounts for fast Ba²⁺ current decay assuming the affinity of the inactivation site on the α₁ subunit to be 100 times lower for Ba²⁺ than Ca²⁺.     

Cooperative regulation of Cav1.2 channels by intracellular Mg2+, the proximal C-terminal EF-hand,and the distal C-terminal domain

L-type Ca²⁺ currents conducted by Ca_v1.2 channels initiate excitation–contraction coupling in cardiac myocytes. Intracellular Mg²⁺ (Mg_i) inhibits the ionic current of Ca_v1.2 channels. Because Mg_i is altered in ischemia and heart failure, its regulation of Ca_v1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mg_i on voltage-dependent inactivation (VDI) of Ca_v1.2 channels using Na⁺ as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca²⁺-dependent inactivation process. We confirmed that increased Mg_i reduces peak ionic currents and increases VDI of Ca_v1.2 channels in ventricular myocytes and in transfected cells when measured with Na⁺ as permeant ion. The increased rate and extent of VDI caused by increased Mg_i were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mg_i to the EF-hand (K_D ≈ 0.9 mM) near the resting level of Mg_i in ventricular myocytes. VDI was more rapid for L-type Ca²⁺ currents in ventricular myocytes than for Ca_v1.2 channels in transfected cells. Coexpression of Ca_vβ_2b subunits and formation of an autoinhibitory complex of truncated Ca_v1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Ca_v1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg_i binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca_v1.2 channels by Mg_i, the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.     

Inactivation of calcium channel current in the calf cardiac Purkinje fiber. Evidence for voltage- and calcium-mediated mechanisms

We have studied the influence of divalent cations on Ca channel current in the calf cardiac Purkinje fiber to determine whether this current inactivates by voltage- or Ca-mediated mechanisms, or by a combination of the two. We measured the reversal (or zero current) potential of the current when Ba, Sr, or Ca were the permeant divalent cations and determined that depletion of charge carrier does not account for time-dependent relaxation of Ca channel current in these preparations. Inactivation of Ca channel current persists when Ba or Sr replaces Ca as the permeant divalent cation, but the voltage dependence of the rate of inactivation is markedly changed. This effect cannot be explained by changes in external surface charge. Instead, we interpret the results as evidence that inactivation is both voltage and Ca dependent. Inactivation of Sr or Ba currents reflects a voltage-dependent process. When Ca is the divalent charge carrier, an additional effect is observed: the rate of inactivation is increased as Ca enters during depolarizing pulses, perhaps because of an additional Ca-dependent mechanism.     

Calcium-dependent Enhancement of Depletion-activated Calcium Current in Jurkat T Lymphocytes

We have obtained evidence that the Ca²⁺-selective current activated by Ca²⁺ store depletion (Ca²⁺ release-activated Ca²⁺ current; I _crac) in Jurkat T lymphocytes is augmented in a time-dependent manner by Ca²⁺ itself. Whole cell patch clamp experiments employed high cytosolic Ca²⁺-buffering conditions to passively deplete Ca²⁺ stores. Rapidly switching to nominally Ca²⁺-free extracellular buffer instantaneously reduced I _crac measured at −100 mV to leak current level. Unexpectedly, readmission of 2 mm Ca²⁺ instantaneously restored only 38 ± 5% (mean ±sem; n = 9) of the full I _crac amplitude. The remainder reappeared in a monotonic time-dependent manner over 10 to 20 sec. Rapid vs. slow intracellular Ca²⁺ chelators did not alter this process, and inorganic I _crac blockers did not regenerate it, arguing against an intracellular site of action. The effect was specific to Ca²⁺: introduction of the permeant ions, Ba²⁺ or Sr²⁺, failed to invoke time-dependent I _crac reappearance. Moreover, equimolar substitution of Ba²⁺ for Ca²⁺ initially produced Ba²⁺ current of similar magnitude to the full Ca²⁺ current, but the Ba²⁺ current decayed monotonically to <50% of its initial amplitude in <20 sec. Conversely, return to Ca²⁺ produced a time-dependent increase in I _crac to its larger Ca²⁺ permeation level. Thus Ca²⁺ appears to selectively promote a reversible transition of I _crac that results in larger current flux, and at least partially explains the selectivity of this current for Ca²⁺ over other divalent ions. Received: 30 August 1995/Revised: 7 November 1995     

Voltage-dependent calcium and potassium conductances in striated muscle fibers from the scorpion,Centruroides sculpturatus

Ionic currents responsible for the action potential in scorpion muscle fibers were characterized using a three-intracellular microelectrode voltage clamp applied at the fiber ends (8–12°C). Large calcium currents (I _Ca) trigger contractile activation in physiological saline (5 mm Ca) but can be studied in the absence of contractile activation in a low Ca saline (2.5 mm). Barium (Ba) ions (1.5–3 mm) support inward current but not contractile activation.Ca conductance kinetics are fast (time constant of 3 msec at 0 mV) and very voltage dependent, with steady-state conductance increasing e-fold in approximately 4 mV. Half-activation occurs at –25 mV. Neither I _Ca nor I _Ba show rapid inactivation, but a slow, voltage-dependent inactivation eliminates I _Ca at voltages positive to –40 mV. Kinetically, scorpion channels are more similar to L-type Ca channels in vertebrate cardiac muscle than to those in skeletal muscle.Outward K currents turn on more slowly and with a longer delay than do Ca currents, and K conductance rises less steeply with voltage (e-fold change in 10 mV; half-maximal level at 0 mV). K channels are blocked by externally applied tetraethylammonium and 3,4 diaminopyridine.This work was supported by a grant from the NIH (NS-17510) to W.F.G. and a NRSA award to T.S. (GM-09921).     

Calcium Channel Current in Rat Dental Pulp Cells

Voltage-gated Ca²⁺ currents in early-passage rat dental pulp cells were studied using whole-cell patch-clamp techniques. With Ba²⁺ as the charge carrier, two prominent inwardly-directed currents, I _f and I _s , were identified in these cells that could be distinguished on the basis of both kinetics and pharmacology. I _f was activated by membrane depolarizations more positive than −30 mV, and displayed fast inactivation kinetics, while I _s was activated by steeper depolarizations and inactivated more slowly. At peak current, time constants of inactivation for I _f and I _s were ∼17 vs.∼631 msec. Both I _f and I _s could be blocked by lanthanum. By contrast, only I _s was sensitive to either Bay-K or nifedipine, a specific agonist and antagonist, respectively, of L-type Ca²⁺ channels. I _s was also blocked by the peptide omega-Conotoxin GVIA. Taken together, results suggested that I _f was mediated by divalent cation flow through voltage-gated T-type Ca²⁺ channels, whereas I _s was mediated by L- and N-type Ca²⁺ channels in the pulp cell membrane. The expression of these prominent, voltage-gated Ca²⁺ channels in a presumptive mineral-inductive phenotype suggests a functional significance vis a vis differentiation of dental pulp cells for the expression and secretion of matrix proteins, and/or formation of reparative dentin itself. Received: 29 November 1999/Revised: 24 April 2000