Pattern of compensatory expression of voltage-dependent Ca2+ channel alpha1 and beta subunits in brain of N-type Ca2+ channel alpha1B subunit gene-deficient mice with a CBA/JN genetic background.

The Ca(2+) channel alpha(1B) subunit is a pore-forming component capable of generating N-type Ca(2+) channel activity. Although the N-type Ca(2+) channel plays a role in a variety of neuronal functions, alpha(1B)-deficient mice with a CBA/JN genetic background show no apparent behavioral or anatomical-histological abnormality, presumably owing to compensation by other Ca(2+) channels. In this study, we examined the mRNA expression of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits in the olfactory bulb, cerebral cortex, hippocampus and cerebellum of alpha(1B)-deficient mice. We found that the mRNA expression levels of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits were the same in the olfactory bulbs of wild, heterozygous and homozygous alpha(1B)-deficient mice. In the cerebral cortex, alpha(1A) mRNA in homozygous alpha(1B)-deficient mice was expressed at a higher level than in wild or heterozygous mice, but no difference in the expression levels of the alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits was found among wild, heterozygous and homozygous mice. In hippocampus and cerebellum, beta(4) mRNA in homozygous alpha(1B)-deficient mice was expressed at a higher level than in wild or heterozygous mice, but no difference in the expression levels of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2) and beta(3) subunits was found among wild, heterozygous and homozygous mice. These results suggest that the compensatory mechanisms differ in different brain regions of alpha(1B)-deficient mice with a CBA/JN genetic background.     

Sequence differences between alpha1C and alpha1S Ca2+ channel subunits reveal structural determinants of a guarded and modulated benzothiazepine receptor

The molecular basis of the Ca2+ channel block by (+)-cis-diltiazem was studied in class A/L-type chimeras and mutant alpha1C-a Ca2+ channels. Chimeras consisted of either rabbit heart (alpha1C-a) or carp skeletal muscle (alpha1S) sequence in transmembrane segments IIIS6, IVS6, and adjacent S5-S6 linkers. Only chimeras containing sequences from alpha1C-a were efficiently blocked by (+)-cis-diltiazem, whereas the phenylalkylamine (-)-gallopamil efficiently blocked both constructs. Carp skeletal muscle and rabbit heart Ca2+ channel alpha1 subunits differ with respect to two nonconserved amino acids in segments IVS6. Transfer of a single leucine (Leu1383, located at the extracellular mouth of the pore) from IVS6 alpha1C-a to IVS6 of alpha1S significantly increased the (+)-cis-diltiazem sensitivity of the corresponding mutant L1383I. An analysis of the role of the two heterologous amino acids in a L-type alpha1 subunit revealed that corresponding amino acids in position 1487 (outer channel mouth) determine recovery of resting Ca2+ channels from block by (+)-cis-diltiazem. The second heterologous amino acid in position 1504 of segment IVS6 (inner channel mouth) was identified as crucial inactivation determinant of L-type Ca2+ channels. This residue simultaneously modulates drug binding during membrane depolarization. Our study provides the first evidence for a guarded and modulated benzothiazepine receptor on L-type channels.     

Two types of Ca2+ currents with different sensitivities to organic Ca2+ channel antagonists in guinea pig pancreatic alpha 2 cells

The possibility that guinea pig pancreatic alpha 2 cells are equipped with more than one type of Ca2+ channel was explored using the patch-electrode voltage-clamp technique. At a holding potential of -100 mV, a slowly developing (tau m approximately 5 ms at -40 mV assuming m2 kinetics) Ca2+ current appeared. This conductance first became detectable at potentials of about -60 mV and reached a maximum amplitude of 50-100 pA between -30 and -20 mV. During long depolarizations, it inactivated completely (tau h approximately 100 ms at -40 mV). Half-maximal steady state inactivation was observed at about -60 mV. A second, more rapidly developing (tau m approximately 2 ms at 0 mV) Ca2+ current was observed during pulses to -40 mV and above. It had a peak amplitude of 150-200 pA between 0 and 10 mV, was less dependent on the holding potential, and inactivated very little, even during long pulses. Both conductances were blocked by Co2+ but were unaffected by tetrodotoxin. The rapidly developing current differed from the slowly developing one in being sensitive to the antagonists D-600 and nifedipine, conducting Ba2+ better than Ca2+, increasing upon exposure to forskolin, and showing time-dependent decay (rundown). These findings indicate that the alpha 2 cells are equipped with two kinds of Ca2+ channels.     

Differential role of the alpha1C subunit tails in regulation of the Cav1.2 channel by membrane potential, beta subunits, and Ca2+ ions

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.     

Structural determinants for functional coupling between the beta and alpha subunits in the Ca2+-activated K+ (BK) channel

High conductance, calcium- and voltage-activated potassium (BK, MaxiK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (beta) subunits. The most remarkable effects of beta1 and beta2 subunits are an increase of the calcium sensitivity and the slow down of channel kinetics. However, the detailed characteristics of channels formed by alpha and beta1 or beta2 are dissimilar, the most remarkable difference being a reduction of the voltage sensitivity in the presence of beta1 but not beta2. Here we reveal the molecular regions in these beta subunits that determine their differential functional coupling with the pore-forming alpha-subunit. We made chimeric constructs between beta1 and beta2 subunits, and BK channels formed by alpha and chimeric beta subunits were expressed in Xenopus laevis oocytes. The electrophysiological characteristics of the resulting channels were determined using the patch clamp technique. Chimeric exchange of the different regions of the beta1 and beta2 subunits demonstrates that the NH3 and COOH termini are the most relevant regions in defining the behavior of either subunit. This strongly suggests that the intracellular domains are crucial for the fine tuning of the effects of these beta subunits. Moreover, the intracellular domains of beta1 are responsible for the reduction of the BK channel voltage dependence. This agrees with previous studies that suggested the intracellular regions of the alpha-subunit to be the target of the modulation by the beta1-subunit.     

Cloning, localization, and functional expression of sodium channel beta1A subunits

Auxiliary beta1 subunits of voltage-gated sodium channels have been shown to be cell adhesion molecules of the Ig superfamily. Co-expression of alpha and beta1 subunits modulates channel gating as well as plasma membrane expression levels. We have cloned, sequenced, and expressed a splice variant of beta1, termed beta1A, that results from an apparent intron retention event. beta1 and beta1A are structurally homologous proteins with type I membrane topology; however, they contain little to no amino acid homology beyond the shared Ig loop region. beta1A mRNA expression is developmentally regulated in rat brain such that it is complementary to beta1. beta1A mRNA is expressed during embryonic development, and then its expression becomes undetectable after birth, concomitant with the onset of beta1 expression. In contrast, beta1A mRNA is expressed in adult adrenal gland and heart. Western blot analysis revealed beta1A protein expression in heart, skeletal muscle, and adrenal gland but not in adult brain or spinal cord. Immunocytochemical analysis of beta1A expression revealed selective expression in brain and spinal cord neurons, with high expression in heart and all dorsal root ganglia neurons. Co-expression of alphaIIA and beta1A subunits in Chinese hamster lung 1610 cells results in a 2.5-fold increase in sodium current density compared with cells expressing alphaIIA alone. This increase in current density reflected two effects of beta1A: 1) an increase in the proportion of cells expressing detectable sodium currents and 2) an increase in the level of functional sodium channels in expressing cells. [(3)H]Saxitoxin binding analysis revealed a 4-fold increase in B(max) with no change in K(D) in cells coexpressing alphaIIA and beta1A compared with cells expressing alphaIIA alone. beta1A-expressing cell lines also revealed subtle differences in sodium channel activation and inactivation. These effects of beta1A subunits on sodium channel function may be physiologically important events in the development of excitable cells.     

Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and gamma subunits. Implications for the structure of cardiac L-type Ca2+ channels.

High threshold L-type Ca2+ channels of skeletal muscle are thought to consist of a complex of alpha 1, alpha 2 delta, beta, and gamma subunits. Expression of the cloned alpha 1 subunit from skeletal and cardiac muscle has established that this protein is the dihydropyridine-sensitive ion-conducting subunit. However, the kinetics of the skeletal muscle alpha 1 alone expressed in mouse L-cells were abnormally slow and were accelerated to within the normal range by coexpression with the skeletal muscle beta subunit. The kinetics of cardiac muscle alpha 1 were also slowed but to a lesser extent and were not altered by coexpression with skeletal muscle alpha 2. We show here that coexpression of the skeletal muscle beta subunit with the cardiac alpha 1 subunit in Xenopus laevis oocytes produced: 1) an increase in the peak voltage-sensitive current, 2) a shift of the peak current-voltage relationship to more hyperpolarized potentials, and 3) an increase in the rate of activation. Coexpression of the skeletal muscle gamma subunit did not have a significant effect on currents elicited by alpha 1. However, when gamma was coexpressed with beta and alpha 1, both peak currents and rates of activation at more negative potentials were increased. These results indicate that rather than simply amplifying expression of alpha 1, heterologous skeletal muscle beta and gamma subunits can modulate the biophysical properties of cardiac alpha 1.     

Cloning of the beta(2a) subunit of the voltage-dependent calcium channel from human heart: cooperative effect of alpha(2)/delta and beta(2a) on the membrane expression of the alpha(1C) subunit

Expression and membrane localization of an epitope-tagged human Ca(2+) channel alpha(1C) subunit were monitored in Xenopus oocytes by confocal microscopy and electrophysiological recording. When alpha(2)/delta and beta(2a) were separately coexpressed with the alpha(1C) subunit, assessment by confocal microscopy showed an 86 and 225% increase of the channel density, respectively. Simultaneous coexpression of alpha(2)/delta and beta(2a) subunits resulted in a cooperative (470%) increase. Electrophysiological measurements performed in parallel revealed that the current augmentation by the alpha(2)/delta subunit is totally attributable to an increase in channel density, whereas the beta(2a) subunit, in addition to increasing channel density, also facilitates channel opening.     

Regulation of Ca-sensitive inactivation of a 1-type Ca2+ channel by specific domains of beta subunits.

Ca2+ channel auxiliary beta subunits have been shown to modulate voltage-dependent inactivation of various types of Ca2+ channels. The beta1 and beta2 subunits, that are differentially expressed with the L-type alpha1 Ca2+ channel subunit in heart, muscle and brain, can specifically modulate the Ca2+-dependent inactivation kinetics. Their expression in Xenopus oocytes with the alpha1C subunit leads, in both cases, to biphasic Ca2+ current decays, the second phase being markedly slowed by expression of the beta2 subunit. Using a series of beta subunit deletion mutants and chimeric constructs of beta1 and beta2 subunits, we show that the inhibitory site located on the amino-terminal region of the beta2a subunit is the major element of this regulation. These results thus suggest that different splice variants of the beta2 subunit can modulate, in a specific way, the Ca2+ entry through L-type Ca2+ channels in different brain or heart regions.     

Cloning and expression of the A2a adenosine receptor from guinea pig brain

A full-length complementary DNA (cDNA) clone encoding the guinea pig brain A₂ adenosine receptor has been isolated by polymerase chain reaction (PCR) and low-stringency-hybridization screening of a guinea pig brain cDNA library. This cDNA contains a long open reading frame encoding a 409-amino acid-residue protein which is highly homologous to the A₂ adenosine receptors previously cloned from other species. Hydrophobicity analysis of the deduced protein sequence reveals seven hydrophobic regions, characteristic of a member of the G-protein-coupled receptor superfamily. Radioligand binding assay and functional (GTPase and cAMP) assays of the receptor, transiently expressed in mammalian cells, demonstrate typical characteristics of the A₂ type adenosine receptor. The messenger RNA (mRNA) of this A₂ receptor is found in the brain, heart, kidney and spleen. Receptor autoradiography with [³H]CGS21680, a specific A₂ agonist, and in situ hybridization with A₂ cRNA probe in guinea pig brain indicate that the receptor is expressed exclusively in the caudate nucleus. The pharmacological profile and anatomical distribution of this receptor indicate that it is of the A_2a subtype. This work represents the first cloning of an A_2a receptor in a rodent species, offers a complete pharmacological characterization of the receptor and provides an anatomical comparison between binding profile and gene expression of the receptor.Abbreviations ADAC adenosine amine congener - BA N⁶-benzyladenosine - bp nucleotide base pair - cAMP cyclic adenosine 3,5-monophosphate - CCPA 2-chloro-N⁶-cyclopentyladenosine - CGS 21680 2-p-(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamido adenosine hydrochloride - CHA N⁶-cyclohexyladenosine - CNS central nervous system - CPA N⁶-cyclohexyladenosine - CNS central nervous system - CPA N⁶-cyclopentyladenosine - CPX 8-cyclopentyl-1,3-dipropylxanthine - DME Dulbecco's modified Eagle's medium - DMPX 3,7-dimethyl-1-propargylxanthine - DPMX 1,3-dipropyl-7-methylxanthine - DPX 1,3-dipropyl-8-(2-amono-4-chlorophenyl)xanthine - FCS fetal calf serum - IBMX 3-isobutyl-1-methylxanthine - KHB Kreb-HEPES buffer - MECA 5-N-methylcarboxamidoadenosine - NECA 5-N-ethylcarboxamidoadenosine - D-PBS Dulbecco's phosphate buffered saline - PCR polymerase chain reaction - R-PIA R(–)-N⁶-(2-phenylisopropyl)adenosine - SSPE sodium chloride-sodium phosphate-EDTA buffer - TM transmembrane domain - XAC xanthine amine congener Special issue dedicated to Dr. Bernard W. Agranoff     

Mechanism of dihydropyridine interaction with critical binding residues of L-type Ca2+ channel alpha 1 subunits

We investigated the mechanism of interaction of individual L-type channel amino acid residues with dihydropyridines within a dihydropyridine-sensitive alpha1A subunit (alpha1A(DHP)). Mutation of individual residues in repeat III and expression in Xenopus oocytes revealed that Thr(1393) is not required for dihydropyridine interaction but that bulky side chains (tyrosine, phenylalanine) in this position sterically inhibit dihydropyridine coordination. In position 1397 a side chain carbonyl group was required for high antagonist sensitivity. Agonist function required the complete amide group of a glutamine residue. Val(1516) and Met(1512) side chains were required for agonist (Val(1516)) and antagonist (Val(1516), Met(1512)) sensitivity. Replacement of Ile(1504) and Ile(1507) by alpha1A phenylalanines was tolerated. Substitution of Thr(1393) by phenylalanine or Val(1516) by alanine introduced voltage dependence of antagonist action into alpha1A(DHP), suggesting that these residues form part of a mechanism mediating voltage dependence of dihydropyridine sensitivity. Our data provide important insight into dihydropyridine binding to alpha1A(DHP) which could facilitate the development of alpha1A-selective modulators. By modulating P/Q-type Ca(2+) channels such drugs could serve as new anti-migraine therapeutics.     

Changes in intracellular Ca2+ concentration induced by L-type Ca2+ channel current in guinea pig gastric myocytes

We investigatedthe relationship between voltage-operatedCa²⁺ channel current and thecorresponding intracellular Ca²⁺concentration([Ca²⁺]_i)change (Ca²⁺ transient) in guineapig gastric myocytes. Fluorescence microspectroscopy was combined withconventional whole cell patch-clamp technique, and fura 2 (80 µM) wasadded to CsCl-rich pipette solution. Step depolarization to 0 mVinduced inward Ca²⁺ current(I_Ca) andconcomitantly raised[Ca²⁺]_i.Both responses were suppressed by nicardipine, an L-typeCa²⁺ channel blocker, and thevoltage dependence of Ca²⁺transient was similar to the current-voltage relation ofI_Ca. When pulseduration was increased by up to 900 ms, peakCa²⁺ transient increased andreached a steady state when stimulation was for longer. The calculatedfast Ca²⁺ buffering capacity(B value), determined as the ratio ofthe time integral ofI_Ca divided bythe amplitude of Ca²⁺ transient,was not significantly increased after depletion of Ca²⁺ stores by the cyclicapplication of caffeine (10 mM) in the presence of ryanodine (4 µM).The addition of cyclopiazonic acid (CPA, 10 µM), a sarco(endo)plasmicreticulum Ca²⁺-ATPase inhibitor,decreased B value by ~20% in areversible manner. When KCl pipette solution was used,Ca²⁺-activatedK⁺ current[I_K(Ca)]was also recorded during step depolarization. CPA sensitivelysuppressed the initial peak and oscillations of I_K(Ca) withirregular effects on Ca²⁺transients. The above results suggest that, in guinea pig gastric myocyte, Ca²⁺ transient is tightlycoupled to I_Caduring depolarization, and global[Ca²⁺]_iis not significantly affected byCa²⁺-inducedCa²⁺ release from sarcoplasmicreticulum during depolarization.

     

Ca2+ stores and Ca2+ entry differentially contribute to the release of IL-1 beta and IL-1 alpha from murine macrophages

Interleukin-1 is a primary mediator of immune responses to injury and infection, but the mechanism of its cellular release is unknown. IL-1 exists as two agonist forms (IL-1 alpha and IL-1 beta) present in the cytosol of activated monocytes/macrophages. IL-1 beta is synthesized as an inactive precursor that lacks a signal sequence, and its trafficking does not use the classical endoplasmic reticulum-Golgi route of secretion. Using primary cultured murine peritoneal macrophages, we demonstrate that P2X7 receptor activation causes release of IL-1 beta and IL-1 alpha via a common pathway, dependent upon the release of Ca(2+) from endoplasmic reticulum stores and caspase-1 activity. Increases in intracellular Ca(2+) alone do not promote IL-1 secretion because a concomitant efflux of K(+) through the plasmalemma is required. In addition, we demonstrate the existence of an alternative pathway for the secretion of IL-1 alpha, independent of P2X7 receptor activation, but dependent upon Ca(2+) influx. The identification of these mechanisms provides insight into the mechanism of IL-1 secretion, and may lead to the identification of targets for the therapeutic modulation of IL-1 action in inflammation.     

Ca(2+)-dependent inactivation of a cloned cardiac Ca2+ channel alpha 1 subunit (alpha 1C) expressed in Xenopus oocytes.

The alpha 1 subunit of cardiac Ca2+ channel, expressed alone or coexpressed with the corresponding beta subunit in Xenopus laevis oocytes, elicits rapidly inactivating Ca2+ currents. The inactivation has the following properties: 1) It is practically absent in external Ba2+; 2) it increases with Ca2+ current amplitudes; 3) it is faster at more negative potentials for comparable Ca2+ current amplitudes; 4) it is independent of channel density; and 5) it does not require the beta subunit. These findings indicate that the Ca2+ binding site responsible for inactivation is encoded in the alpha 1 subunit and suggest that it is located near the inner channel mouth but outside the membrane electric field.