Adolfo Cavalié

A novel capacitative calcium entry channel expressed in excitable cells

In addition to voltage‐gated calcium influx, capacitative calcium entry (CCE) represents a major pathway for calcium entry into the cell. Here we report the structure, expression and functional properties of a novel CCE channel, TRP5. This channel is a member of a new subfamily of mammalian homologues of the Drosophila transient receptor potential (TRP) protein, now comprising TRP5 (also CCE2) and the structurally related CCE1 (also TRP4). Like TRP4, TRP5 forms ion channels mainly permeable for Ca2+ which are not active under resting conditions but can be activated by manoeuvres known to deplete intracellular calcium stores. Accordingly, dialysis of TRP5‐expressing cells with inositol‐(1,4,5)‐trisphosphate evokes inward rectifying currents which reversed polarity at potentials more positive than +30 mV. Ca2+ store depletion with thapsigargin induced TRP5‐mediated calcium entry dependent on the concentration of extracellular calcium, as seen by dual wavelength fura‐2 fluorescence ratio measurements. TRP5 transcripts are expressed almost exclusively in brain, where they are present in mitral cells of the olfactory bulb, in lateral cerebellar nuclei and, together with TRP4 transcripts, in CA1 pyramidal neurons of the hippocampus, indicating the presence of CCE channels in excitable cells and their participation in neuronal calcium homeostasis.

A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL

Intracellular Ca2+ signalling evoked by Ca2+ mobilizing agonists, like angiotensin II in the adrenal gland, involves the activation of inositol(1,4,5)trisphosphate(InsP3)‐mediated Ca2+ release from internal stores followed by activation of a Ca2+ influx termed capacitative calcium entry. Here we report the amino acid sequence of a functional capacitative Ca2+ entry (CCE) channel that supports inward Ca2+ currents in the range of the cell resting potential. The expressed CCE channel opens upon depletion of Ca2+ stores by InsP3 or thapsigargin, suggesting that the newly identified channel supports the CCE coupled to InsP3 signalling.

TRP4 (CCE1) protein is part of native calcium release-activated Ca2+-like channels in adrenal cells.

Mammalian TRP proteins have been implicated to function as ion channel subunits responsible for agonist-induced Ca2+ entry. To date, TRP proteins have been extensively studied by heterologous expression giving rise to diverse channel properties and activation mechanisms including store-operated mechanisms. However, the molecular structure and the functional properties of native TRP channels still remain elusive. Here we analyze the properties of TRP4 (CCE1) channels in their native environment and characterize TRP expression patterns and store-operated calcium currents that are endogenous to bovine adrenal cells. We show by Northern blot analysis, immunoblots, and immunohistochemistry thatTRP4 transcripts and TRP4 protein are present in the adrenal cortex but absent in the medulla. Correspondingly, bovine adrenal cortex cells express TRP4 abundantly. The only otherTRP transcript found at considerable levels wasTRP1, whereas TRP2, TRP3, TRP5(CCE2), andTRP6 were not detectable. Depletion of calcium stores with inositol 1,4,5-trisphosphate or thapsigargin activates store-operated ion channels in adrenal cells. These channels closely resemble calcium release-activated Ca2+ (CRAC) channels. Expression of trp4(CCE1) cDNA in antisense orientation significantly reduces both, the endogenous CRAC-like currents and the amount of native TRP4 protein. These results demonstrate that TRP4 contributes essentially to the formation of native CRAC-like channels in adrenal cells.

Phenotype of a recombinant store-operated channel: highly selective permeation of Ca2+

1 Genes related to trp (transient receptor potential) are proposed to encode store‐operated channels. We examined the ionic permeation of recombinant channels formed by stable and transient expression of the TRP homologue bCCE1 in Chinese hamster ovary (CHO) cells (CHO(CCE1)) and rat basophilic leukaemia (RBL) cells, respectively. 2 Store‐operated currents were activated in CHO(CCE1) cells by internal dialysis of IP3 under strong buffering of intracellular Ca2+. The action of IP3 was mimicked by thapsigargin but not by IP4. 3 With extracellular Ca2+, Na+ and Mg2+, the store‐operated currents of CHO(CCE1) rectified inwardly in the presence of internal Cs+. Outward currents were not detected below +80 mV. Identical currents were recorded with external Ba2+ and also with no external Na+ and Mg2+. In the absence of external Mg2+, the inward currents showed an anomalous mole fraction behaviour between Ca2+ and Na+. Half‐maximal inhibition of Na+ currents was observed with ≈100 nM and full block with 2‐5 μM external Ca2+. 4 In the parental CHO(‐) cells, IP3 dialysis evoked inward currents that also displayed anomalous mole fraction behaviour between Ca2+ and Na+. However, half‐maximal block of Na+ currents required 5 times higher Ca2+ concentrations in CHO(‐) cells. Additionally, the density of Ca2+ and Na+ currents at ‐80 mV was 5 and 2 times larger in CHO(CCE1) cells, respectively. 5 In RBL cells, dialysis of IP3 evoked store‐operated currents that showed 1.4‐fold larger densities at ‐80 mV in cells expressing bCCE1. 6 The enhanced density of store‐operated currents in CHO(CCE1) cells and in bCCE1‐transfected RBL cells probably reflects the phenotype of CCE1. These results suggest a highly selective permeation of Ca2+ through recombinant channels formed by CCE1 either alone or in combination with endogenous channel proteins.

Temperature-induced transitory and steady-state changes in the calcium current of guinea pig ventricular myocytes

ICa was recorded in quinea pig ventricular myocytes using the whole-cell voltage-clamp technique. The shape of the I–V relation was unaffected by temperature (21–37°C) but there were large changes in ICa amplitude and time course. Steady-state responses indicated Q10 s of 2.96±0.14 (amplitude), 2.52±0.13 (time to peak), and 2.82±0.28 (T1/2 inactivation) (mean ± SD, n=6). Quick changes in temperature (T1/2<30 s) induced pronounced deviations from the steady-state Q10 relations (early depression, compensatory overshoot). Thus, cardiac ICa differs from other currents in having a high amplitude-Q10 and an oscillatory response to rapid temperature changes.

Fast and slow gating behaviour of single calcium channels in cardiac cells

Abstract(1)The Ca-channel gating behaviour during steady and stepwise depolarization was examined in recordings of single Ca-channel activity from cell-attached membrane patches of single ventricular cells isolated enzymatically from hearts of adult guinea pigs. The single-channel recordings were performed by means of the improved patch-clamp technique (Hamill et al. 1981) with 90 mM Ba in the pipettes.(2)Upon step depolarization, two types of current records were regularly observed in the ensembles: (1) traces with Ca-channel activity (in the form of closely-spaced brief pulses of inward current with a unitary amplitude) of various length, and (2) blank sweeps without any detectable single-channel opening. The records with Ca-channel activity show a distinct tendency for openings to occur towards the beginning of the clamp pulse, followed by long periods of silence. The blank sweeps seem to reflect a condition or conditions where the Ca channel is unavailable for opening. The corresponding ensemble mean currentI(t) displayed a rapid rising phase to its peak followed by a slow decay.(3)During steady depolarization, kinetic analysis of the distributions of all open and shut lifetimes revealed a monoexponential probability density distribution function of all open times. By contrast, more than two exponential terms were required for an accurate description of the frequency distribution of all shut lifetimes. Corresponding to the two well-separated fast closed time components, individual Ca-channel openings were grouped into bursts of openings. The bursting behaviour reflected fast gating transitions and was related to the fluctuations of the Ca channel between two short-lived closed states and one open state. This fast gating was terminated by the entrance of the Ca channel into at least one long-lived closed state, exit from which was slow in comparison to the rapid cycling. As consequence, bursts of openings were further grouped together in clusters of bursts, the cluster behaviour being related to slow gating transitions in the kinetics of the Ca channel.(4)The biphasic frequency distribution of the first latencies (resulting from the transit through the two short-lived shut states, before the open state is entered) superimposed on the first time derivative of the rising phase of the ensemble mean current,I(t), upon step depolarization. The time constant of the monoexponential distribution function of all cluster lifetimes matched the declining phase ofI(t) during maintained depolarization. Thus, the decrease of the probability of channel opening and the resulting decline ofI(t) seemed to be due to a transition of the Ca channel into the long-lived third class of shut state(s).(5)The responsiveness of the Ca channel in a series of trials decreased at positive holding potentials with a sigmoidal dependence on the potential of the conditioning depolarization due to an increasing number of blank single-channel current records within the ensembles. Traces with channel openings as well as blank sweeps tended to form sequences of consecutive single-channel current records upon conditioning depolarization. The Ca channel was only activatable, if a command pulse was applied during the occupancy of either the open state or the two short-lived shut states. If the long-lived shut state(s) was already occupied at the conditioning potential preceding the step depolarization, the Ca channel was unavailable for opening and a blank sweep was observed upon the voltage pulse.(6)A quantitative patch-to-patch variability in Ca-channel gating behaviour was detected. It was interpreted as the statistical deviation from the average kinetic behaviour of a single population of Ca channels.(7)The total time course of the ensemble mean currentI(t) was reconstructed by a convolution of the first latency and cluster lifetime distribution functions. The peak amplitude ofI(t) was mainly determined by the steady-state occupancy of the activatable states of the Ca channel.(8)Under comparable experimental conditions (90 mM external Ba), the pooled average behaviour of individual Ca channels in different membrane patches was the same as the bulk behaviour of all the Ca channels in the cardiac cell.(9)The fast and slow Ca-channel gating transitions are discussed in terms of a channel-state model where, according to lifetimes and transition rates, the respective channel states are divided into two subsets.

TRPC5 Is a Ca2+-activated Channel Functionally Coupled to Ca2+-selective Ion Channels

TRPC5 forms non-selective cation channels. Here we studied the role of internal Ca2+ in the activation of murine TRPC5 heterologously expressed in human embryonic kidney cells. Cell dialysis with various Ca2+ concentrations (Ca2+i) revealed a dose-dependent activation of TRPC5 channels by internal Ca2+ with EC50 of 635.1 and 358.2 nm at negative and positive membrane potentials, respectively. Stepwise increases of Ca2+i induced by photolysis of caged Ca2+ showed that the Ca2+ activation of TRPC5 channels follows a rapid exponential time course with a time constant of 8.6 ± 0.2 ms at Ca2+i below 10 μm, suggesting that the action of internal Ca2+ is a primary mechanism in the activation of TRPC5 channels. A second slow activation phase with a time to peak of 1.4 ± 0.1 s was also observed at Ca2+i above 10 μm. In support of a Ca2+-activation mechanism, the thapsigargin-induced release of Ca2+ from internal stores activated TRPC5 channels transiently, and the subsequent Ca2+ entry produced a sustained TRPC5 activation, which in turn supported a long-lasting membrane depolarization. By co-expressing STIM1 plus ORAI1 or the α1C and β2 subunits of L-type Ca2+ channels, we found that Ca2+ entry through either calcium-release-activated-calcium or voltage-dependent Ca2+ channels is sufficient for TRPC5 channel activation. The Ca2+ entry activated TRPC5 channels under buffering of internal Ca2+ with EGTA but not with BAPTA. Our data support the hypothesis that TRPC5 forms Ca2+-activated cation channels that are functionally coupled to Ca2+-selective ion channels through local Ca2+ increases beneath the plasma membrane.

BiP‐mediated closing of the Sec61 channel limits Ca2+ leakage from the ER

In mammalian cells, signal peptide‐dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic protein‐conducting channel, the Sec61 complex. Previous work has characterized the Sec61 channel as a potential ER Ca2+ leak channel and identified calmodulin as limiting Ca2+ leakage in a Ca2+‐dependent manner by binding to an IQ motif in the cytosolic aminoterminus of Sec61α. Here, we manipulated the concentration of the ER lumenal chaperone BiP in cells in different ways and used live cell Ca2+ imaging to monitor the effects of reduced levels of BiP on ER Ca2+ leakage. Regardless of how the BiP concentration was lowered, the absence of available BiP led to increased Ca2+ leakage via the Sec61 complex. When we replaced wild‐type Sec61α with mutant Sec61αY344H in the same model cell, however, Ca2+ leakage from the ER increased and was no longer affected by manipulation of the BiP concentration. Thus, BiP limits ER Ca2+ leakage through the Sec61 complex by binding to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344.

Voltage-dependent properties of macroscopic and elementary calcium channel currents in guinea pig ventricular myocytes

Whole-cell Ca channel currents were recorded from guinea pig ventricular myocytes that were internally perfused with Cs solution and bathed in solutions containing 3.6 mM Ca, 3.6 mM Ba or 90 mM Ba (34° C). Single Ca channel currents were recorded from cell-attached membrane patches of similar myocytes; the patch pipettes contained a 90 mM Ba solution. 1. Although the shape of the whole-cellI–V relation was independent of the bathing solution, this was not the case with the location of the inward current maximum (Vpeak);Vpeak in 90 mM Ba was about 30 mV positive toVpeak in 3.6 mM Ba. 2. The activation and inactivation of whole-cell currents were voltage dependent. Compared to the voltage dependencies in 3.6 mM Ba, those in 90 mM Ba were shifted by about 30 mV to the right, suggesting a neutralization of surface charges. 3. Observations compatible with the ion permeation model proposed by Hess and Tsien (1984) included (a) a depression of current during Ca/Ba solution exchange, (b) a high divalent to monovalent ion permeability, and (c) rectification of the outward limb of theI–V relation. 4. Estimated current densities atVpeak were similar for myocytes in 3.6 mM Ca and 3.6 mM Ba, and about 10 times larger in 90 mM Ba. 5. Average currents (I*) calculated from ensembles of records of single Ca channel current had voltage-dependent time courses resembling those of whole-cellIBa (90 mM). 6. Single-channelI*–V relations were superimposable on whole-cellI–V curves suggesting that voltage-dependent single-channel parameters (probability of opening, elementary current amplitude) can be related to the voltage-dependent macroscopic current parameters (activation, instantaneousI–V relation) when scaled by channel number. 7. The density of Ca channels in myocytes was calculated from whole-cellIBa (90 mM) and average current through single channels. The outcome, 3–5 channels/μm2, agrees with two other recent estimates (Tsien et al. 1983; Lux and Brown 1984). However, it is difficult to reconcile with the much lower density that one would forecast from the frequency of functional channel observation in myocyte membrane patches (Pelzer et al. 1985c).

Pain Perception in Mice Lacking the β3 Subunit of Voltage-activated Calcium Channels

The importance of voltage-activated calcium channels in pain processing has been suggested by the spinal antinociceptive action of blockers of N- and P/Q-type calcium channels as well as by gene targeting of the α1B subunit (N-type). The accessory β3 subunits of calcium channels are preferentially associated with the α1B subunit in neurones. Here we show that deletion of the β3 subunit by gene targeting affects strongly the pain processing of mutant mice. We pinpoint this defect in the pain-related behavior and ascending pain pathways of the spinal cordin vivo and at the level of calcium channel currents and proteins in single dorsal root ganglion neurones in vitro. The pain induced by chemical inflammation is preferentially damped by deletion of β3 subunits, whereas responses to acute thermal and mechanical harmful stimuli are reduced moderately or not at all, respectively. The defect results in a weak wind-up of spinal cord activity during intense afferent nerve stimulation. The molecular mechanism responsible for the phenotype was traced to low expression of N-type calcium channels (α1B) and functional alterations of calcium channel currents in neurones projecting to the spinal cord.