Ca2+ entry-independent effects of L-type Ca2+ channel modulators on Ca2+ sparks in ventricular myocytes

During the cardiac action potential, Ca²⁺ entry through dyhidropyridine receptor L-type Ca²⁺ channels (DHPRs) activates ryanodine receptors (RyRs) Ca²⁺-release channels, resulting in massive Ca²⁺ mobilization from the sarcoplasmic reticulum (SR). This global Ca²⁺ release arises from spatiotemporal summation of many localized elementary Ca²⁺-release events, Ca²⁺ sparks. We tested whether DHPRs modulate Ca²⁺sparks in a Ca²⁺ entry-independent manner. Negative modulation by DHPR of RyRs via physical interactions is accepted in resting skeletal muscle but remains controversial in the heart. Ca²⁺ sparks were studied in cat cardiac myocytes permeabilized with saponin or internally perfused via a patch pipette. Bathing and pipette solutions contained low Ca²⁺ (100 nM). Under these conditions, Ca²⁺ sparks were detected with a stable frequency of 3–5 sparks·s^–1·100 µm^–1. The DHPR blockers nifedipine, nimodipine, FS-2, and calciseptine decreased spark frequency, whereas the DHPR agonists Bay-K8644 and FPL-64176 increased it. None of these agents altered the spatiotemporal characteristics of Ca²⁺ sparks. The DHPR modulators were also without effect on SR Ca²⁺ load (caffeine-induced Ca²⁺ transients) or sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) activity (Ca²⁺ loading rates of isolated SR microsomes) and did not change cardiac RyR channel gating (planar lipid bilayer experiments). In summary, DHPR modulators affected spark frequency in the absence of DHPR-mediated Ca²⁺ entry. This action could not be attributed to a direct action of DHPR modulators on SERCA or RyRs. Our results suggest that the activity of RyR Ca²⁺-release units in ventricular myocytes is modulated by Ca²⁺ entry-independent conformational changes in neighboring DHPRs. exitation-contraction coupling; ryanodine receptor; sarco(endo)plasmic reticulum Ca²⁺-ATPase; dihydropyridine receptor; sarcoplasmic reticulum     

L-type Ca2+ channels in Ca2+ channelopathies

Voltage-gated L-type Ca2+ channels (LTCCs) mediate depolarization-induced Ca2+ entry in electrically excitable cells, including muscle cells, neurons, and endocrine and sensory cells. In this review we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within pore-forming alpha1 subunits causing incomplete congenital stationary night blindness, malignant hyperthermia sensitivity or hypokalemic periodic paralysis. However, studies in mice revealed that LTCC dysfunction also contributes to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Ca(v)2.1 alpha1 in tottering mice. Ca2+ channelopathies provide exciting molecular tools to elucidate the contribution of different LTCC isoforms to human diseases.     

Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L-type calcium channels

Elevated intracellular Ca2+ triggers inactivation of L-type calcium channels, providing negative Ca2+ feedback in many cells. Ca2+ binding to the main alpha1c channel subunit has been widely proposed to initiate such Ca2+ -dependent inactivation. Here, we find that overexpression of mutant, Ca2+ -insensitive calmodulin (CaM) ablates Ca2+ -dependent inactivation in a "dominant-negative" manner. This result demonstrates that CaM is the actual Ca2+ sensor for inactivation and suggests that CaM is constitutively tethered to the channel complex. Inactivation is likely to occur via Ca2+ -dependent interaction of tethered CaM with an IQ-like motif on the carboxyl tail of alpha1c. CaM also binds to analogous IQ regions of N-, P/Q-, and R-type calcium channels, suggesting that CaM-mediated effects may be widespread in the calcium channel family.     

Contribution of spontaneous L-type Ca2+ channel activation to the genesis of Ca2+ sparks in resting cardiac myocytes

Ca2+ sparks are the elementary events of intracellular Ca2+ release from the sar-coplasmic reticulum in cardiac myocytes. In order to investigate whether spontaneous L-type Ca2+ channel activation contributes to the genesis of spontaneous Ca2+ sparks, we used confocal laser scanning microscopy and fluo-4 to visualize local Ca2+ sparks in intact rat ventricular myocytes. In the presence of 0.2 mmol/L CdCI2 which inhibits spontaneous L-type Ca2+ channel activation, the rate of occurrence of spontaneous Ca2+ sparks was halved from 4.20 to 2.04 events/(100 μm·s), with temporal and spatial properties of individual Ca2+ sparks unchanged. Analysis of the Cd2+-sensitive spark production revealed an open probability of-10-5 for L-type channels at the rest membrane potentials (-80 mV). Thus, infrequent and stochastic openings of sarcolemmal L-type Ca2+ channels in resting heart cells contribute significantly to the production of spontaneous Ca2+ sparks.     

Potentiated L-type Ca2+ channels rectify

Strong depolarization and dihydropyridine agonists potentiate inward currents through native L-type Ca2+ channels, but the effect on outward currents is less clear due to the small size of these currents. Here, we examined potentiation of wild-type alpha1C and two constructs bearing mutations in conserved glutamates in the pore regions of repeats II and IV (E2A/E4A-alpha1C) or repeat III (E3K-alpha1C). With 10 mM Ca2+ in the bath and 110 mM Cs+ in the pipette, these mutated channels, expressed in dysgenic myotubes, produced both inward and outward currents of substantial amplitude. For both the wild-type and mutated channels, we observed strong inward rectification of potentiation: strong depolarization had little effect on outward tail currents but caused the inward tail currents to be larger and to decay more slowly. Similarly, exposure to DHP agonist increased the amplitude of inward currents and decreased the amplitude of outward currents through both E2A/E4A-alpha1C and E3K-alpha1C. As in the absence of drug, strong depolarization in the presence of dihydropyridine agonist had little effect on outward tail currents but increased the amplitude and slowed the decay of inward tail currents. We tested whether cytoplasmic Mg2+ functions as the blocking particle responsible for the rectification of potentiated L-type Ca2+ channels. However, even after complete removal of cytoplasmic Mg2+, (-)BayK 8644 still potentiated inward current and partially blocked outward current via E2A/E4A-alpha1C. Although zero Mg2+ did not reveal potentiation of outward current by DHP agonist, it did have two striking effects, (a) a strong suppression of decay of both inward and outward currents via E2A/E4A-alpha1C and (b) a nearly complete elimination of depolarization-induced potentiation of inward tail currents. These results can be explained by postulating that potentiation exposes a binding site in the pore to which an intracellular blocking particle can bind and produce inward rectification of the potentiated channels.     

Interactions between H+ and Ca2+ near cardiac L-type calcium channels: evidence for independent channel-associated binding sites.

Monovalent and divalent ions are known to affect voltage-gated ion channels by the screening of, and/or binding to, negative charges located on the surface of cell membranes within the vicinity of the channel protein. In this investigation, we studied gating shifts of cardiac L-type calcium channels induced by extracellular H+ and Ca2+ to determine whether these cations interact at independent or competitive binding sites. At constant pHo (7.4), Cao-induced gating shifts begin to approach a maximum value (approximately equal to 17 mV) at concentrations of extracellular calcium of > or = 40 mM. A fraction of the calcium-dependent gating shift could be titrated with an effective pKa = 6.9 indicating common and competitive access to H+ and Ca2+ ions for at least one binding site. However, if pHo is lowered when Cao is > or = 40 mM, additional shifts in gating are measured, suggesting a subpopulation of sites to which Ca2+ and H+ bind independently. The interdependence of L-channel gating shifts and Cao and pHo was well described by the predictions of surface potential theory in which two sets of binding sites are postulated; site 1 (pKa = 5.5) is accessible only to H+ ions and site 2 (pKa = 6.9) is accessible to both Ca2+ and H+ ions. Theoretical computations generated with this model are consistent with previously determined data, in which interactions between these two cations were not studied, in addition to the present experiments in which interactions were systematically probed.     

Temperature-sensitive intracellular Mg2+ block of L-type Ca2+ channels in cardiac myocytes

We examined the concentration-dependent blocking effects of intracellular Mg2+ on L-type Ca2+ channels in cardiac myocytes using the whole cell patch-clamp technique. The increase of L-type Ca2+ channel current (I(Ca)) (due to relief of Mg2+ block) occurred in two temporal phases. The rapid phase (runup) transiently appeared early (<5 min) in dialysis of the low-Mg2+ solution; the slow phase began later in dialysis (>10 min). Runup was not blocked by intracellular GTP (GTP(i)). The late phase of the I(Ca) increase (late I(Ca)) was suppressed by GTP(i) (0.4 mM) and was observed in myocytes of the guinea pig or frog at higher (32 or 24 degrees C, respectively) rather than lower temperatures (24 or 17.5 degrees C, respectively). At pMg = 6.0, raising the temperature from 24 to 32 degrees C evoked late I(Ca) with a Q10 of 14.5. Restoring the temperature to 24 degrees C decreased I(Ca) with a Q10 of only 2.4. The marked difference in the Q10 values indicated that late I(Ca) (pMg = 5-6) is an irreversible phenomenon. Phosphorylation suppressed the intracellular [Mg2+] dependency of late I(Ca). This effect of phosphorylation together with the inhibitory action of GTP(i) on Mg2+-dependent blocking of I(Ca) are common properties of mammalian and amphibian cardiomyocytes.     

Large currents generate cardiac Ca2+ sparks

Previous models of cardiac Ca2+ sparks have assumed that Ca2+ currents through the Ca2+ release units (CRUs) were approximately 1-2 pA, producing sparks with peak fluorescence ratio (F/F(0)) of approximately 2.0 and a full-width at half maximum (FWHM) of approximately 1 microm. Here, we present actual Ca2+ sparks with peak F/F(0) of >6 and a FWHM of approximately 2 microm, and a mathematical model of such sparks, the main feature of which is a much larger underlying Ca2+ current. Assuming infinite reaction rates and no endogenous buffers, we obtain a lower bound of approximately 11 pA needed to generate a Ca2+ spark with FWHM of 2 microm. Under realistic conditions, the CRU current must be approximately 20 pA to generate a 2- microm Ca2+)spark. For currents > or =5 pA, the computed spark amplitudes (F/F(0)) are large (approximately 6-12 depending on buffer model). We considered several factors that might produce sparks with FWHM approximately 2 microm without using large currents. Possible protein-dye interactions increased the FWHM slightly. Hypothetical Ca2+ "quarks" had little effect, as did blurring of sparks by the confocal microscope. A clusters of CRUs, each producing 10 pA simultaneously, can produce sparks with FWHM approximately 2 microm. We conclude that cardiac Ca2+ sparks are significantly larger in peak amplitude than previously thought, that such large Ca2+ sparks are consistent with the measured FWHM of approximately 2 microm, and that the underlying Ca2+ current is in the range of 10-20 pA.     

Galpha q inhibits cardiac L-type Ca2+ channels through phosphatidylinositol 3-kinase

Cardiac myocyte contractility is initiated by Ca2+ entry through the voltage-dependent L-type Ca2+ channel (LTCC). To study the effect of Galpha q on the cardiac LTCC, we utilized two transgenic mouse lines that selectively express inducible Galpha q-estrogen receptor hormone-binding domain fusion proteins (Galpha qQ209L-hbER or Galpha qQ209L-AA-hbER) in cardiac myocytes. Both of these proteins inhibit phosphatidylinositol (PI) 3-kinase (PI3K) signaling, but Galpha qQ209L-AA-hbER cannot activate the canonical Galpha q effector phospholipase Cbeta (PLCbeta). L-type Ca2+ current (I(Ca,L)) density measured by whole-cell patch clamping was reduced by more than 50% in myocytes from both Galpha q animals as compared with wild-type cells, suggesting that inhibition of the LTCC by Galpha q does not require PLCbeta. To investigate the role of PI3K in this inhibitory effect, I(Ca,L) was measured in the presence of various phosphoinositides infused through the patch pipette. Infusion of PI 3,4,5-trisphosphate (PI(3,4,5)P3) into wild-type myocytes did not affect I(Ca,L), but it fully restored I(Ca,L) density in both Galpha q transgenic myocytes to wild-type levels. By contrast, PI 4,5-bisphosphate (PI(4,5)P2) or PI 3,5-bisphosphate had no effect. Infusion with p110beta/p85alpha or p110gamma PI3K in the presence of PI(4,5)P2 also restored I(Ca,L) density to wild-type levels. Last, infusion of either PTEN, a PI(3,4,5)P3 phosphatase, or the pleckstrin homology domain of Grp1, which sequesters PI(3,4,5)P3, reduced the peak I(Ca,L) density in wild-type myocytes by approximately 30%. Taken together, these results strongly suggest that the inhibitory effect of Galpha q on the cardiac LTCC is mediated by inhibition of PI3K.     

Extremely slow inactivation of the ion channels formed by transfected α2 of L-type Ca2+ channelsof L-type Ca2+ channels

Barium currents through ion channels formed by α1-subunit of L-type Ca²⁺ channel (I _α1) were recorded from cultured chinese hamster ovary (CHO) cells. The cells were stably transfected with either a cardiac or a smooth muscle (SM) variant of α1-subunit. TheI _α1 in both cases exhibited similar fast voltage-dependent activation kinetics and slow apparent inactivation kinetics. With 10 mM Ba²⁺ in the bath solution,I _α1 was activated at potentials more positive than −40 mV, peaked between 0 and +10 mV, and reversed at about +50 mV. In addition to slow apparent inactivation of inward current, both subunits provided an extremely slow voltage-dependent inactivation at potentials more positive than −100 mV, with half-maximum inactivation at −43.4 mV for cardiac and −41.4 mV for SM α1-subunits. The onset of inactivation as well as recovery from this process were within a time range of minutes. The voltage dependence of steady-state inactivation could be fitted by the sum of two Boltzmann's equations with slope factors of about 12 mV and 5 mV. A less sloped component has its midpoints at −75.6 and −63.7 mV, and a steeper component has its midpoints at −42.8 and −37.7 mV for cardiac and SM α1-subunits, respectively. Relative contribution of the steeper component was higher in both subunits (0.86 and 0.66 for cardiac and SM subunits, respectively). For comparison, the inactivation curves for 5-sec-long conditioning prepulses could be fitted by single Boltzmann's distribution with a 20 mV more positive midpoint and a slope factor of about 13 mV. In contrast to the steady-state inactivation curves, they showed considerable overlap with the steady-state activation curve. Our results reflect functional consequences of known sequence differences between α1-subunits of the cardiac and SM L-type Ca²⁺ channels and could be used in structural modeling of Ca²⁺ channel gating. In addition, they show that depolarization-induced window current has a transient nature and decays with the development of extremely slow inactivation. This is the first demonstration that slow inactivation of the L-type Ca²⁺ channel is an intrinsic property of its α1-subunits.     

The effects of chronic depolarization on L-type 1,4-dihydropyridine-sensitive, voltage-dependent Ca2+ channels in chick neural retina and rat cardiac cells.

Chick neural retina cells contain functional L-type voltage-dependent Ca2+ channels sensitive to 1,4-dihydropyridines. To investigate the effects of chronic depolarization, cells were grown in medium containing elevated K+. After 4-h to 4-day treatments with elevated K+ (12-73 mM), there was a concentration-dependent decrease in high affinity [3H]PN200-110 binding. Saturation analysis of cells treated for 4 days with 40 mM K+ showed a reduction in maximum ligand binding with no change in affinity. Control and experimental Bmax values were 70.7 +/- 6.4 and 42.2 +/- 4.5 fmol/mg protein, respectively, and control and experimental KD values were 70.2 +/- 7.4 and 68.6 +/- 7.4 x 10(-12) M. The effect of chronic depolarization was time-dependent, reversible, and without effect on cellular protein content. Reduction in 45Ca2+ uptake following chronic depolarization correlated well with the reduction in [3H]PN200-110 binding. The calcium ionophore A23187, 10(-6) M for 24 h, also decreased the binding site density. The calcium channel antagonist D600 had no effect alone on [3H]PN200-110 binding; however, D600 blocked the down-regulation of calcium channels induced by chronic depolarization. The mechanism for Ca2+ channel down-regulation may involve calcium entry, since the effect was blocked by D600 and mimicked by the calcium ionophore A23187. Chronic depolarization with either elevated K+ or veratridine, or chronic treatment with A23187 had no effect on calcium channels in rat neonatal ventricular myocytes, although these cells express functional channels of the 1,4-dihydropyridine-sensitive class.(ABSTRACT TRUNCATED AT 250 WORDS)     

The beta subunit increases Ca2+ currents and gating charge movements of human cardiac L-type Ca2+ channels.

The properties of the gating currents (nonlinear charge movements) of human cardiac L-type Ca2- channels and their relationship to the activation of the Ca2+ channel (ionic) currents were studied using a mammalian expression system. Cloned human cardiac alpha1 + rabbit alpha 2 subunits or human cardiac alpha 1 + rabbit alpha 2 + human beta 3 subunits were transiently expressed in HEK293 cells. The maximum Ca2+ current density increased from -3.9 +/- 0.9 pA/pF for the alpha 1 + alpha 2 subunits to -11.6 +/- 2.2 pA/pF for alpha 1 + alpha 2 + beta 3 subunits. Calcium channel gating currents were recorded after the addition of 5 mM Co2+, using a -P/5 protocol. The maximum nonlinear charge movement (Qmax) increased from 2.5 +/- 0.3 nC/muF for alpha 1 + alpha 2 subunit to 12.1 +/- 0.3 nC/muF for alpha 1 + alpha 2 + beta 3 subunit expression. The QON was equal to the QOFF for both subunit combinations. The QON-Vm data were fit by a sum of two Boltzmann expressions and ranged over more negative potentials, as compared with the voltage dependence for activation of the Ca2+ conductance. We conclude that 1) the beta subunit increases the number of functional alpha 1 subunits expressed in the plasma membrane of these cells and 2) the voltage-dependent activation of the human cardiac L-type calcium channel involves the movements of at least two nonidentical and functionally distinct gating structures.     

A region of calpastatin domain L that reprimes cardiac L-type Ca2+ channels

Calpastatin, an endogenous inhibitor of calpain, is composed of domain L and four repetitive homologous domains 1-4. Domains 1-4 inhibit calpain, whereas domain L partially reprimes L-type Ca2+ channels for voltage-gated activation. In the present study, the effects on Ca2+ channel activity of four isoforms and a series of fragments of calpastatin domain L were investigated in guinea-pig ventricular myocytes with the patch-clamp method. With one exception, all the isoforms and fragment peptides that contained amino acid residues 54-64 of domain L reprimed the Ca2+ channels to comparable levels (9-15% of control activity) to those observed previously with a full-length form of calpastatin. These results suggest that the region containing amino acid residues 54-64 (EGKPKEHTEPK) is responsible for the Ca2+ channel repriming function of calpastatin domain L.     

The role of stochastic and modal gating of cardiac L-type Ca2+ channels on early after-depolarizations

Certain signaling events that promote L-type Ca2+ channel (LCC) phosphorylation, such as beta-adrenergic stimulation or an increased expression of Ca(2+)/calmodulin-dependent protein kinase II, promote mode 2 gating of LCCs. Experimental data suggest the hypothesis that these events increase the likelihood of early after-depolarizations (EADs). We test this hypothesis using an ionic model of the canine ventricular myocyte incorporating stochastic gating of LCCs and ryanodine-sensitive calcium release channels. The model is extended to describe myocyte responses to the beta-adrenergic agonist isoproterenol. Results demonstrate that in the presence of isoproterenol the random opening of a small number of LCCs gating in mode 2 during the plateau phase of the action potential (AP) can trigger EADs. EADs occur randomly, where the likelihood of these events increases as a function of the fraction of LCCs gating in mode 2. Fluctuations of the L-type Ca2+ current during the AP plateau lead to variability in AP duration. Consequently, prolonged APs are occasionally observed and exhibit an increased likelihood of EAD formation. These results suggest a novel stochastic mechanism, whereby phosphorylation-induced changes in LCC gating properties contribute to EAD generation.     

Reconstitution of local Ca2+ signaling between cardiac L-type Ca2+ channels and ryanodine receptors: insights into regulation by FKBP12.6

Ca⁺-induced Ca²⁺ release (CICR) in the heart involves local Ca²⁺ signaling between sarcolemmal L-type Ca²⁺ channels (dihydropyridine receptors, DHPRs) and type 2 ryanodine receptors (RyR2s) in the sarcoplasmic reticulum (SR). We reconstituted cardiac-like CICR by expressing a cardiac dihydropyridine-insensitive (T1066Y/Q1070M) ₁-subunit (1C_YM) and RyR2 in myotubes derived from RyR1-knockout (dyspedic) mice. Myotubes expressing 1C_YM and RyR2 were vesiculated and exhibited spontaneous Ca²⁺ oscillations that resulted in chaotic and uncontrolled contractions. Coexpression of FKBP12.6 (but not FKBP12.0) with 1C_YM and RyR2 eliminated vesiculations and reduced the percentage of myotubes exhibiting uncontrolled global Ca²⁺ oscillations (63% and 13% of cells exhibited oscillations in the absence and presence of FKBP12.6, respectively). 1C_YM/RyR2/FKBP12.6-expressing myotubes exhibited robust and rapid electrically evoked Ca²⁺ transients that required extracellular Ca²⁺. Depolarization-induced Ca²⁺ release in 1C_YM/RyR2/FKBP12.6-expressing myotubes exhibited a bell-shaped voltage dependence that was fourfold larger than that of myotubes expressing 1C_YM alone (maximal fluorescence change was 2.10 ± 0.39 and 0.54 ± 0.07, respectively), despite similar Ca²⁺ current densities. In addition, the gain of CICR in 1C_YM/RyR2/FKBP12.6-expressing myotubes exhibited a nonlinear voltage dependence, being considerably larger at threshold potentials. We used this molecular model of local _1C-RyR2 signaling to assess the ability of FKBP12.6 to inhibit spontaneous Ca²⁺ release via a phosphomimetic mutation in RyR2 (S2808D). Electrically evoked Ca²⁺ release and the incidence of spontaneous Ca²⁺ oscillations did not differ in wild-type RyR2- and S2808D-expressing myotubes over a wide range of FKBP12.6 expression. Thus a negative charge at S2808 does not alter in situ regulation of RyR2 by FKBP12.6. heart failure; dihydropyridine receptor; excitation-contraction coupling     

Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain.     

Role of Cav1.2 L-type Ca2+ channels in vascular tone: effects of nifedipine and Mg2+

Ca(2+) entry via L-type voltage-gated Ca(2+) channels (LVGCs) is a key factor in generating myogenic tone (MT), as dihydropyridines (DHPs) and other LVGC blockers, including Mg(2+), markedly reduce MT. Recent reports suggest, however, that elevated external Mg(2+) concentration and DHPs may also inhibit other Ca(2+)-entry pathways. Here, we explore the contribution of LVGCs to MT in intact, pressurized mesenteric small arteries using mutant mice (DHP(R/R)) expressing functional but DHP-insensitive Ca(v)1.2 channels. In wild-type (WT), but not DHP(R/R), mouse arteries, nifedipine (0.3-1.0 microM) markedly reduced MT and vasoconstriction induced by high external K(+) concentrations ([K(+)](o)), a measure of LVGC-mediated Ca(2+) entry. Blocking MT and high [K(+)](o)-induced vasoconstriction by <1 microM nifedipine in WT but not in DHP(R/R) arteries implies that Ca(2+) entry via Ca(v)1.2 LVGCs is obligatory for MT and that nifedipine inhibits MT exclusively by blocking LVGCs. We also examined the effects of Mg(2+) on MT and LVGCs. High external Mg(2+) concentration (10 mM) blocked MT, slowed the high [K(+)](o)-induced vasoconstrictions, and decreased their amplitude in WT and DHP(R/R) arteries. To verify that these effects of Mg(2+) are due to block of LVGCs, we characterized the effects of extracellular and intracellular Mg(2+) on LVGC currents in isolated mesenteric artery myocytes. DHP-sensitive LVGC currents are inhibited by both external and internal Mg(2+). The results indicate that Mg(2+) relaxes MT by inhibiting Ca(2+) influx through LVGCs. These data provide new information about the central role of Ca(v)1.2 LVGCs in generating and maintaining MT in mouse mesenteric small arteries.     

Ca(2+)-dependent inactivation of cardiac L-type Ca2+ channels does not affect their voltage sensor

Inactivation of currents carried by Ba2+ and Ca2+, as well as intramembrane charge movement from L-type Ca2+ channels were studied in guinea pig ventricular myocytes using the whole-cell patch clamp technique. Prolonged (2 s) conditioning depolarization caused substantial reduction of charge movement between -70 and 10 mV (charge 1, or charge from noninactivated channels). In parallel, the charge mobile between -70 and -150 mV (charge 2, or charge from inactivated channels) was increased. The availability of charge 2 depended on the conditioning pulse voltage as the sum of two Boltzmann components. One component had a central voltage of -75 mV and a magnitude of 1.7 nC/microF. It presumably is the charge movement (charge 2) from Na+ channels. The other component, with a central voltage of approximately - 30 mV and a magnitude of 3.5 nC/microF, is the charge 2 of L-type Ca2+ channels. The sum of charge 1 and charge 2 was conserved after different conditioning pulses. The difference between the voltage dependence of the activation of L-type Ca2+ channels (half-activation voltage, V, of approximately -20 mV) and that of charge 2 (V of -100 mV) made it possible to record the ionic currents through Ca2+ channels and charge 2 in the same solution. In an external solution with Ba2+ as sole metal the maximum available charge 2 of L-type Ca2+ channels was 10-15% greater than that in a Ca(2+)-containing solution. External Cd2+ caused 20-30% reduction of charge 2 both from Na+ and L-type Ca2+ channels. Voltage- and Ca(2+)-dependent inactivation phenomena were compared with a double pulse protocol in cells perfused with an internal solution of low calcium buffering capacity. As the conditioning pulse voltage increased, inactivation monitored with the second pulse went through a minimum at about 0 mV, the voltage at which conditioning current had its maximum. Charge 2, recorded in parallel, did not show any increase associated with calcium entry. Two alternative interpretations of these observations are: (a) that Ca(2+)- dependent inactivation does not alter the voltage sensor, and (b) that inactivation affects the voltage sensor, but only in the small fraction of channels that open, and the effect goes undetected. A model of channel gating that assumes the first possibility is shown to account fully for the experimental results. Thus, extracellular divalent cations modulate voltage-dependent inactivation of the Ca2+ channel. Intracellular Ca2+ instead, appears to cause inactivation of the channel without affecting its voltage sensor.