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Voltage‐gated Ca2+ channels are key transducers of cellular excitability and participate in several crucial physiological responses. In vertebrates, 10 Ca2+ channel genes, grouped in 3 families (CaV1, CaV2 and CaV3), have been described and characterized. Insects possess only one member of each family. These genes have been isolated in a limited number of species and very few have been characterized although, in addition to their crucial role, they may represent a collateral target for neurotoxic insecticides. We have isolated the 3 genes coding for the 3 Ca2+ channels expressed in Apis mellifera. This work provides the first detailed characterization of the honeybee T-type CaV3 Ca2+ channel and demonstrates the low toxicity of inhibiting this channel. Comparing Ca2+ currents recorded in bee neurons and myocytes with Ca2+ currents recorded in Xenopus oocytes expressing the honeybee CaV3 gene suggests native expression in bee muscle cells only. High‐voltage activated Ca2+ channels could be recorded in the somata of different cultured bee neurons. These functional data were confirmed by in situ hybridization, immunolocalization and in vivo analysis of the effects of a CaV3 inhibitor. The biophysical and pharmacological characterization and the tissue distribution of CaV3 suggest a role in honeybee muscle function.

The β subunits of voltage-gated Ca 2+ channels are known to be regulators of the channels' gating properties. Here we report a striking additional function of a β subunit. Screening of chicken cochlear and brain cDNA libraries identified β 4c , a short splice variant of the β 4 subunit. Although β 4c occurs together with the longer isoforms β 4a or β 4b in the brain, eye, heart, and lung, the cochlea expresses exclusively β 4c . The association of β 4c with the Ca 2+ -channel α 1 subunit has slight but significant effects on the kinetics of channel activation and inactivation. Yeast two-hybrid and biochemical assays revealed that β 4c interacts directly with the chromo shadow domain of chromobox protein 2/heterochromatin protein 1γ (CHCB2/HP1γ), a nuclear protein involved in gene silencing and transcriptional regulation. Coexpression of this protein specifically recruits β 4c to the nuclei of mammalian cells. Furthermore, β 4c but not β 4a dramatically attenuates the gene-silencing activity of chromobox protein 2/heterochromatin protein 1γ. The β 4c subunit is therefore a multifunctional protein that not only constitutes a portion of the Ca 2+ channel but also regulates gene transcription.

1. High-voltage-activated Ca2+ current (ICa) waveforms in adult rat ventricular myocytes comprise two components, referred to here as ICa(fc) and ICa(sc) to denote the fast and slow components, respectively, of ICa decay. At all test potentials, the two time constants of ICa decay, tau fc and tau sc, differ by approximately an order of magnitude. Neither tau fc nor tau sc varies appreciably with test potential, however, suggesting that current inactivation is not markedly voltage dependent. 2. Current activation at all test potentials follows a sigmoidal time course and is best described by a power function with n = 4. Deactivation of the currents, examined following variable length depolarizations to various test potentials, however, follows a single exponential time course. In addition, the kinetics of activation and deactivation of ICa(fc) and ICa(sc) are indistinguishable. 3. Although both begin to activate at approximately -30 mV, the voltage dependences of ICa(fc) and ICa(sc) are distinct: ICa(fc) peaks at -10 mV and ICa(sc) peaks at +10 mV. 4. The relative amplitudes of ICa(fc) and ICa(sc) vary with the holding potential from which the currents are evoked and with the frequency of current activation: hyperpolarized holding potentials and low stimulation frequencies reveal preferential activation of ICa(fc), whereas depolarized holding potentials and high stimulation frequencies potentiate ICa(sc). In addition, the observed voltage- and frequency-dependent changes in ICa(fc) and ICa(sc) amplitudes are reciprocal. 5. The apparent voltage dependences of steady-state inactivation of ICa(fc) and ICa(sc) are also distinct. ICa(fc) is reduced to approximately 50% of its maximal amplitude at -45 mV, whereas ICa(sc) is approximately 50% inactivated at -30 mV. 6. Recovery of ICa(peak) from steady-state inactivation follows a complex time course. Following inactivation at -10 mV, ICa(peak) recovers at -90 mV to its maximal value over a biexponential time course; ICa(peak) then decreases over the next several seconds to a steady-state level. 7. The time course of recovery from steady-state inactivation of ICa(fc) at -90 mV is best described by the sum of two exponentials; the two time constants of recovery differ by approximately a factor of 25. ICa(sc), in contrast, recovers rapidly and over a single exponential time course to its maximal value. When the recovery time at -90 mV is increased, however, ICa(sc) amplitude decreases slowly and over a single exponential time course to a steady-state level.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Co-injection of RNA synthesized from cloned neuronal acetylcholine receptor (nAChR) subunits (alpha 4 and beta 2) in Xenopus oocytes produced functional receptors. In macroscopic voltage-clamp experiments, the agonist-induced current exhibited a strong inward rectification. 2. Voltage jumps from +50 mV to more negative potentials produced relaxations of the agonist-induced current with a single exponential time course. The relaxation rate constant was only weakly voltage dependent. 3. At the single-channel level, three conductances were recorded of 12, 22 and 34 pS. Their burst durations were similar and varied only weakly with voltage (e-fold for 120 to 370 mV), consistent with the poorly voltage-dependent relaxation rate constants. However, the burst durations were less than 10 ms, or less than 1/5 the value expected from voltage-jump relaxations. 4. Hexamethonium (Hex, 0.5 to 8 microM) inhibited the agonist-induced current and produced voltage-jump relaxations characterized by a rapid conductance increase and a slower conductance decrease. Analysis of these relaxations suggested that the Hex-receptor interaction is open-channel blockade characterized by a forward binding rate of 1 x 10(7) M-1 s-1 and a dissociation rate constant of about 25 s-1. 5. For the relaxations produced by QX222, the slowest phase was a conductance increase, suggesting that the dissociation rate constant for QX222 is 10-30-fold greater than for Hex. 6. Hex but not QX222 produced an additional use-dependent blockade that was manifest during repetitive hyperpolarizing pulses. 7. With mouse muscle ACh receptors expressed in oocytes, the blockade by Hex did not depend strongly on voltage. Neither Hex nor QX222 produced appreciable use-dependent block on muscle ACh receptors. 8. Of the four conditions studied (neuronal and muscle receptors, Hex and QX222), only the blockade of the neuronal AChR by Hex is characterized by a residence time longer than the normal open time. 9. It is concluded that the modest differences in primary amino acid sequence between muscle and neuronal receptors lead to profound changes in their interactions with channels blockers.

We have been examining the interaction of a local anesthetic derivative, QX-222, with the ion channel pore of the muscle AChR, using a combination of mutagenesis, oocyte expression, and electrophysiology. Single channel recording, together with macroscopic voltage-jump relaxations, provides a measure of the residence time of the open channel blocker within the pore. We have found systematic changes in the apparent affinity of the open channel for QX-222 following amino acid substitutions in the proposed M2 transmembrane helix of each of the four subunits of the AChR. Assigning the number 1' to the residue at the cytoplasmic end of the M2 helix, positions 2',6',10',14', and 18' are modeled as forming the lining of the pore. Polar to nonpolar substitutions at 6' decrease QX-222 residence time, while the opposite effect is seen at position 10'. Nonpolar to polar substitutions have the converse effect. The distance between the aromatic and quaternary amine moieties of QX-222 corresponds almost exactly to the repeat distance of an alpha helix. This structural feature is common to many local anesthetic drugs. We propose a model for the binding of QX-222 within the ion channel of the AChR that is consistent with these observations.

Stargazin or [gamma]2, the product of the gene mutated in the stargazer mouse, is a homologue of the [gamma]1 protein, an accessory subunit of the skeletal muscle L-type Ca2+ channel. [gamma]2 is selectively expressed in the brain, and considered to be a putative neuronal Ca2+ channel subunit based mainly on homology to [gamma]1. Two new members of the [gamma] family expressed in the brain have recently been identified: [gamma]3 and [gamma]4. We have co-expressed, in Xenopus oocytes, the human [gamma]2, [gamma]3 and [gamma]4 subunits with the P/Q-type (Ca(V)2.1) Ca2+ channel and different regulatory subunits ([alpha]2-[delta]; [beta]1, [beta]2, [beta]3 or [beta]4). Subcellular distribution of the [gamma] subunits confirmed their membrane localization. Ba2+ currents, recorded using two-electrode voltage clamp, showed that the effects of the [gamma] subunits on the electrophysiological properties of the channel are, most of the time, minor. However, a fraction of the oocytes expressing [beta] subunits displayed an unusual slow-inactivating Ba2+ current. Expression of both [beta] and [gamma] subunits increased the appearance of the slow-inactivating current. Our data support a role for the [gamma] subunit as a brain Ca2+ channel modulatory subunit and suggest that [beta] and [gamma] subunits are involved in a switch between two regulatory modes of the P/Q-type channel inactivation.

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.

Voltage-dependent facilitation of L-type Ca2+ channels is an important regulatory mechanism by which excitable cells modulate Ca2+ entry during a train of action potentials. Expression of the alpha1 and beta subunits of the alpha1C Ca2+ channel is necessary and sufficient to reproduce this kind of facilitation in Xenopus oocytes. Here we show that, by expressing the alpha1C together with different beta subunits in oocytes, the beta1, beta3 and beta4, but not the beta2 subunits are permissive for Ca2+ channel facilitation. The poor facilitation observed in rat ventricular cells, together with the presence of the beta2 subunit mRNA, suggest that beta2 may be the beta subunit associated with functional cardiac L-type Ca2+ channels.

To investigate the effect of acetylcholine receptor (AChR) mutations on neuromuscular transmission and to develop a model for the human neuromuscular disease, the slow-channel syndrome, we generated transgenic mice with abnormal AChRs using a delta subunit with a mutation in the ion channel domain. In three transgenic lines, nerve-evoked end-plate currents and spontaneous miniature end-plate currents (MEPCs) had prolonged decay phases and MEPC amplitudes were reduced by 33%. Single nerve stimuli elicited repetitive compound muscle action potentials in vivo. Transgenic mice were abnormally sensitive to the neuromuscular blocker, curare. These observations demonstrate that we can predictably alter AChR function, synaptic responses, and muscle fiber excitation in vivo by overexpressing subunits containing well-defined mutations. Furthermore these data support the hypothesis that the electrophysiological findings in the neuromuscular disorder, the slow-channel syndrome, are due to mutant AChRs.

Calcium entry into excitable cells through voltage-gated calcium channels can be influenced by both the rate and pattern of action potentials. We report here that a cloned neuronal alpha 1C L-type calcium channel can be facilitated by positive pre-depolarization. Both calcium and barium were effective as charge carriers in eliciting voltage-dependent facilitation. The induction of facilitation was shown to be independent of intracellular calcium levels, G-protein interaction and the level of phosphatase activity. Facilitation was reduced by the injection of inhibitors of protein kinase A and required the coexpression of a calcium channel beta subunit. In contrast, three neuronal non-L-type calcium channels, alpha 1A, alpha 1B and alpha 1E, were not subject to voltage-dependent facilitation when coexpressed with a beta subunit. The results indicate that the mechanism of neuronal L-type calcium channel facilitation involves the interaction of alpha 1 and beta subunits and is dependent on protein kinase A activity. The selective voltage-dependent modulation of L-type calcium channels is likely to play an important role in neuronal physiology and plasticity.

The primary structure of a calcium channel beta subunit (beta M) from housefly (Musca domestica) has been deduced by cDNA cloning and sequence analysis. The open reading frame encodes a 441-amino acid polypeptide with a calculated molecular mass of 48,755 Da. Whole-mount in situ hybridization indicates that beta M mRNA is predominantly expressed in neuronal tissues. Transcription of beta M mRNA is evident from stage 13/14 of embryogenesis up to adulthood. Different expression patterns of splice variants were found in larvae and in adult fly heads. Amino acid identity between beta M and mammalian beta subunits is lower (66-68%) than within mammalian beta subunits (74-80%). Calculation of a phylogenetic tree indicates that beta M is an ancestral form of the four distinct beta subunit gene products identified in mammalian tissues so far. Despite these sequence differences, beta M is able to enhance endogenous calcium channel activity in Xenopus laevis oocytes as well as dihydropyridine binding to membranes from COS 7 cells transfected with rabbit heart alpha 1 cDNA in the same manner as was previously shown for mammalian beta subunits.

The nicotinic acetylcholine receptor can be expressed in Xenopus oocytes by injection of in vitro synthesized RNA for the alpha, beta, gamma, and delta mouse muscle subunits. However, detectable responses can also be obtained by injection of alpha, beta, and delta subunit RNA only. The receptors expressed in this case (gamma-less receptors) share many of the properties of the normal receptor, including relaxation time constants, Hill slope, and relative permeability for Na+, K+, Cs+, and Tris+. The major single-channel conductances of alpha beta gamma delta and alpha beta delta receptors are similar (34.2 +/- 2.9 and 38.5 +/- 0.6 pS, respectively) but clearly different from the major conductances seen after the combined injection of alpha beta delta mouse subunit RNA and Xenopus gamma subunit RNA. Mutations in the second transmembrane segment of the alpha and beta subunits, known to affect open time and blockade by QX-222, are equally effective in the gamma-less receptor. These data strongly suggest that the gamma-less receptor has the same pore diameter as the normal receptor and that alpha, beta, and delta subunits participate in its formation. Injection of alpha beta gamma delta well as alpha beta delta RNA produced additional subconductance states of around 25 pS. The low conductance state was sensitive to mutations introduced in the alpha or beta subunits with or without the gamma subunit, indicating that this channel did not need the gamma subunits but required at least the alpha and beta subunits to be produced. Injection of alpha beta delta and the adult-type epsilon subunit RNA gave rise to channels with conductances of 35 and 55 pS when the stoichiometry of the injection was 2:1:1:1, but only the 55-pS channel was recorded when the epsilon subunit RNA concentration was increased by 10-fold (stoichiometry of 2:1:1:10). The gamma-less receptor can thus be expressed even when the adult epsilon subunit is present. Whether gamma-less receptors are expressed at normal adult neuromuscular junctions remains unknown.

The binding site for an open-channel blocker, QX-222, at mouse muscle nicotinic acetylcholine receptors was probed using site-directed mutagenesis, oocyte expression, and electrophysiological analysis. The proposed cytoplasmic end of the M2 transmembrane helix is termed position 1'. At position 10' (alpha S252, beta T263, gamma A261, delta A266), Ala residues yield stronger and longer binding of QX-222 than Ser or Thr residues. These effects are opposite and roughly equal (30%-50% per mutation) to previously reported effects at position 6'. The polar end of an anesthetic molecule seems to bind to the position 6' OH groups, which provide a water-like region; the nonpolar moiety is near position 10' and binds more strongly in a nonpolar environment. Interactions with adjacent OH-rich turns of an amphiphilic helix may explain the widespread blocking effects of local anesthetics at the conduction pore of ion channels.

Les ions calcium sont impliques dans le developpement et le controle de nombreuses fonctions cellulaires allant du couplage excitation-contraction musculaire, a la liberation d'hormones ou de neurotransmetteurs, a l'activation d'enzymes ou a l'expression de certains genes. L'entree rapide de calcium dans les cellules excitables est reglee principalement par des macromolecules de la membrane plasmique, les canaux calciques dependants du potentiel, qui constituent une classe heterogene de proteines sensibles aux variations du potentiel membranaire. Les travaux d'identification et de caracterisation de plusieurs types de canaux calciques ont revele leur grande diversite et permettent de mieux comprendre comment les cellules utilisent le signal calcique pour controler des fonctions physiologiques tres variees

Site-directed mutagenesis and expression in Xenopus oocytes were used to study acetylcholine receptors in which serine residues (i) were replaced by alanines (alpha, delta subunits) or (ii) replaced a phenylalanine (beta subunit) at a postulated polar site within the M2 transmembrane helix. As the number of serines decreased, there were decreases in the residence time and consequently the equilibrium binding affinity of QX-222, a quaternary ammonium anesthetic derivative thought to bind within the open channel. Receptors with three serine-to-alanine mutations also displayed a selective decrease in outward single-channel currents. Both the direction of this rectification and the voltage dependence of QX-222 blockade suggest that the residues mutated are within the aqueous pore of the receptor and near its cytoplasmic (inner) surface.

THE entry of calcium ions into cells through voltage-activated Ca2+ channels in the plasma membrane triggers many important cellular processes. The activity of these channels is regulated by several hormones and neuretransmitters, as well as intracellular messengers such as Ca2+ itself (for examples, see refs 1–9). In cardiac muscle, myoplasmic Ca2+ has been proposed to potentiate Ca2+ influx1,7–9, although a direct effect of Ca2+ on these channels has not yet been demonstrated. Photosensitive 'caged-Ca2+ molecules such as nitr-5, however, provide powerful tools for investigating possible regulatory roles of Ca2+ on the functioning of Ca2+ channels10,11. Because its affinity for Ca2+ is reduced by irradiation, nitr-5 can be loaded into cells and induced to release Ca2+ with a flash of light10. By using this technique we found that the elevation of intracellular Ca2+ concentration directly augmented Ca2+-channel currents in isolated cardiac muscle cells from both frog and guinea pig. The time course of the current potentiation was similar to that seen with β-adrenergic stimulation. Thus Ca2+ may work through a similar pathway, involving phosphorylation of a regulatory Ca2+-channel protein. This mechanism is probably important for the accumulation of Ca2+ and the amplification of the contractile response in cardiac muscle, and may have a role in other excitable cells.

The effects of nifedipine and nicardipine, 2 dihydropyridines (DHP) used in the treatment of cardiovascular disorders, were compared in frog atrial fibers. Rapid photolysis of nifedipine with a single UV flash (1-ms duration) reversed the block, allowing comparison of effects of both drugs on the same preparation, and manipulation of the calcium channel on a millisecond timescale. The results show that inhibition of the action potential (AP) and slow inward current (Isi) is more pronounced with nifedipine than with nicardipine. Concentration-effect relationships confirm that nicardipine (IC50 = 1 microM) is less potent than nifedipine (IC50 = 0.2 microM) in blocking cardiac calcium channels. Both DHP block calcium channels in the closed state at the resting potential, inducing a large tonic block (in the absence of stimulation). An additional phasic block can be observed with nifedipine and nicardipine. A slight voltage dependence to the block is observed for both DHP, their effects being enhanced depolarization holding potentials. Rapid unblocking of calcium channels by a single light flash, presented during the decay phase of Isi, reveals a higher affinity of DHP for inactivated channels. This effect is most pronounced when inactivation is slowed by using Ba++, Sr++, or Na+ ions as the current carriers. Open channel block is also suggested. The mechanism of DHP action on calcium channels can be described according to the "modulated receptor hypothesis". These DHP exhibit an additional nonspecific effect on potassium channels. It is concluded that nicardipine is a less potent Ca++ antagonist than nifedipine in atrial fibers and that the reduction of delayed potassium current, which occurs in a similar range of concentrations to the blockade of Isi, could also be involved in its therapeutic effects.