Low voltage-activated Ca 2ϩ channels play important roles in pacing neuronal firing and producing network oscillations, such as those that occur during sleep and epilepsy. Here we describe the cloning and expression of the third member of the T-type family, ␣1I or Ca v T.3, from rat brain. Northern analysis indicated that it is predominantly expressed in brain. Expression of the cloned channel in either Xenopus oocytes or stably transfected human embryonic kidney-293 cells revealed novel gating properties. We compared these electrophysiological properties to those of the cloned T-type channels ␣1G and ␣1H and to the high voltage-activated channels formed by ␣1E␤ 3 . The ␣1I channels opened after small depolarizations of the membrane similar to ␣1G and ␣1H but at more depolarized potentials. The kinetics of activation and inactivation were dramatically slower, which allows the channel to act as a Ca 2ϩ injector. In oocytes, the kinetics were even slower, suggesting that components of the expression system modulate its gating properties. Steady-state inactivation occurred at higher potentials than any of the other T channels, endowing the channel with a substantial window current. The ␣1I channel could still be classified as T-type by virtue of its criss-crossing kinetics, its slow deactivation (tail current), and its small (11 pS) conductance in 110 mM Ba 2ϩ solutions. Based on its brain distribution and novel gating properties, we suggest that ␣1I plays important roles in determining the electroresponsiveness of neurons, and hence, may be a novel drug target.

Expression of rat alpha1G, human alpha1H and rat alpha1I subunits of voltage-activated Ca2 + channels in HEK-293 cells yields robust Ca2 + inward currents with 1.25 mM Ca2 + as the charge carrier. Both similarities and marked differences are found between their biophysical properties. Currents induced by expression of alpha1G show the fastest activation and inactivation kinetics. The alpha1H and alpha1I currents activate and inactivate up to 1.5- and 5-fold slower, respectively. No differences in the voltage dependence of steady state inactivation are detected. Currents induced by expression of alpha1G and alpha1H deactivate with time constants of up to 6 ms at a test potential of - 80 mV, but currents induced by alpha1I deactivate about three-fold faster. Recovery from short-term inactivation is more than three-fold slower for currents induced by alpha1H and alpha1I in comparison to alpha1G. In contrast to these characteristics, reactivation after long-term inactivation was fastest for currents arising from expression of alpha1I and slowest in cells expressing alpha1H calcium channels. The calcium inward current induced by expression of alpha1I is increased by positive prepulses while currents induced by alpha1H and alpha1G show little ( < 5%) or no facilitation. The data thus provide a characteristic fingerprint of each channel's activity, which may allow correlation of the alpha1G, alpha1H and alpha1I induced currents with their in vivo counterparts.

Concerted activation of different voltage-gated Ca 2+ channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type Ca V 2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in Ca V 2.3-knockout (Ca V 2.3 -/-) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca 2+ channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological Ca V 2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca 2+ signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single Ca V 2.3 -/-β cells. Ca V 2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the Ca V 2.3 -/-mouse. We propose a specific role for Ca V 2.3 Ca 2+ channels in second-phase insulin release, that of mediating the Ca 2+ entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation. IntroductionSystemic glucose tolerance is orchestrated by the regulated release of insulin and glucagon from the β and α cells of the pancreatic islets of Langerhans. The α and β cells are electrically excitable and use electrical signals to couple changes in blood glucose concentration to stimulation or inhibition of hormone release. In both cell types, influx of extracellular Ca 2+ through voltage-gated Ca 2+ channels with resultant elevation of intracellular Ca 2+ concentration ([Ca 2+ ] i ) triggers exocytosis of the hormone-containing secretory granules. Like other electrically excitable cells, both α and β cells contain several types of voltage-gated Ca 2+ channel (1, 2). Assigning physiological functions to the respective Ca 2+ channels is central to the understanding of electrical and secretory activities in these cells.Voltage-gated Ca 2+ channels are divided into 3 subfamilies: (a) L-type high voltage-activated (HVA) Ca 2+ channel family that comprises the Ca V 1.1, 1.2, 1.3, and 1.4 channels and is inhibited by dihydropyridines (DHPs) (1, 3, 4); (b) non-L-type HVA channels Ca V 2.1 (P/Q-type), 2.2 (N-type), and 2.3 (R-type) that are sensitive to ω-agatoxin IVA and ω-conotoxin GVIA and SNX482, respectively (1, 4, 5); and (c) the low voltage-activated (LVA) T-type Ca 2+ channel family (Ca V 3.1, 3.2, and 3.3). The latter subtype differs electrophysiologically from the HVA Ca 2+ channels in opening transiently already upon modest depolarization (6, 7) and fulfilling important roles in pacemaker cells (8).

Systemic acidosis has detrimental effects on the skeleton, and local acidosis coincides with bone destruction in inflammatory and metastatic diseases. Acidification dramatically enhances osteoclastic resorption, although the underlying mechanism has remained elusive. We investigated the effect of acidosis on the osteoclastogenic transcription factor NFATc1, which upon dephosphorylation translocates from the cytoplasm to nuclei.

Concerted activation of different voltage-gated Ca 2+ channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type Ca V 2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in Ca V 2.3-knockout (Ca V 2.3 -/-) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca 2+ channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological Ca V 2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca 2+ signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single Ca V 2.3 -/-β cells. Ca V 2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the Ca V 2.3 -/-mouse. We propose a specific role for Ca V 2.3 Ca 2+ channels in second-phase insulin release, that of mediating the Ca 2+ entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation. IntroductionSystemic glucose tolerance is orchestrated by the regulated release of insulin and glucagon from the β and α cells of the pancreatic islets of Langerhans. The α and β cells are electrically excitable and use electrical signals to couple changes in blood glucose concentration to stimulation or inhibition of hormone release. In both cell types, influx of extracellular Ca 2+ through voltage-gated Ca 2+ channels with resultant elevation of intracellular Ca 2+ concentration ([Ca 2+ ] i ) triggers exocytosis of the hormone-containing secretory granules. Like other electrically excitable cells, both α and β cells contain several types of voltage-gated Ca 2+ channel (1, 2). Assigning physiological functions to the respective Ca 2+ channels is central to the understanding of electrical and secretory activities in these cells.Voltage-gated Ca 2+ channels are divided into 3 subfamilies: (a) L-type high voltage-activated (HVA) Ca 2+ channel family that comprises the Ca V 1.1, 1.2, 1.3, and 1.4 channels and is inhibited by dihydropyridines (DHPs) (1, 3, 4); (b) non-L-type HVA channels Ca V 2.1 (P/Q-type), 2.2 (N-type), and 2.3 (R-type) that are sensitive to ω-agatoxin IVA and ω-conotoxin GVIA and SNX482, respectively (1, 4, 5); and (c) the low voltage-activated (LVA) T-type Ca 2+ channel family (Ca V 3.1, 3.2, and 3.3). The latter subtype differs electrophysiologically from the HVA Ca 2+ channels in opening transiently already upon modest depolarization (6, 7) and fulfilling important roles in pacemaker cells (8).

Voltage-operated Ca2+ channels are heteromultimeric proteins. Their structural diversity is caused by several genes encoding homologous subunits and by alternative splicing of single transcripts. Isoforms of alpha1 subunits, which contain the ion conducting pore, have been deduced from each of the six cDNA sequences cloned so far from different species. The isoforms predicted for the alpha1E subunit are structurally related to the primary sequence of the amino terminus, the centre of the subunit (II-III loop), and the carboxy terminus. Mouse and human alpha1E transcripts have been analysed by reverse transcription-polymerase chain reaction and by sequencing of amplified fragments. For the II-III loop three different alpha1E cDNA fragments are amplified from mouse and human brain, showing that isoforms originally predicted from sequence alignment of different species are expressed in a single one. Both predicted alpha1E cDNA fragments of the carboxy terminus are identified in vivo. Two different alpha1E constructs, referring to the major structural difference in the carboxy terminus, were stably transfected in HEK293 cells. The biophysical properties of these cells were compared in order to evaluate the importance in vitro of the carboxy terminal insertion found in vivo. The wild-type alpha1E subunit showed properties, typical for a high-voltage activated Ca2+ channel. The deletion of 43 amino acid residues at the carboxy terminus does not cause significant differences in the current density and the basic biophysical properties. However, a functional difference is suggested, as in embryonic stem cells, differentiated in vitro to neuronal cells, the pattern of transcripts indicative for different alpha1E isoforms changes during development. In human cerebellum the longer alpha1E isoform is expressed predominantly. Although, it has not been possible to assign functional differences to the two alpha1E constructs tested in vitro, the expression pattern of the structurally related isoforms may have functional importance in vivo.

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The expression of Ca 2ϩ channel A1E isoforms has been analyzed in different cell lines, embryoid bodies and tissues. The comparison of the different cloned A1E cDNA sequences led to the prediction of A1E splice variants. Transcripts of two cloned A1E isoforms, which are discriminated by a carboxy terminal 129-bp sequence, have been detected in different cell lines and tissues. Transcripts of the shorter A1E isoform have been assigned to the rat cerebrum and to neuron-like cells from in vitro, differentiated embryonic stem cells. The shorter isoform is the major transcript amplified from total RNA by reverse transcription (RT)-PCR and visualized on the protein level by Western blotting with common and isoformspecific antibodies. Transcripts of the longer A1E isoform have been identified in mouse, rat and human cerebellum, in in vitro, differentiated embryoid bodies, in the insulinoma cell lines INS-1 (rat) and βTC-3 (mouse), in the pituitary cell line AtT-20 (mouse) when grown in 5 mM glucose, and in islets of Langerhans (rat) and kidney (rat and human). The detection of different isoforms of A1E in cell lines and tissues shows that the wide expression of A1E has to be specified by identifying the corresponding isoforms in each tissue. In islets of Langerhans and in kidney, a distinct isoform called A1Ee has been determined by RT-PCR, while in cerebellum a set of different A1E structures has been detected, which might reflect the functional heterogeneity of cerebellar neurons. The tissue-specific expression of different isoforms might be related to specific functions, which are not yet known, but the expression of the new isoform A1Ee in islets of Langerhans and kidney leads to the suggestion that A1E might be involved in the modulation of the Ca 2ϩ -mediated hormone secretion.Keywords : A1E isoforms ; Ca 2ϩ channels ; islets of Langerhans; kidney; single-cell RT-PCR.Low and high voltage-gated Ca 2ϩ channels are subdivided, according to their threshold of activation and their different single channel conductances [1]. The pore-forming subunit of the first low voltage-activated T-type Ca 2ϩ channel has been cloned and functionally expressed recently as A1G [2].The group of six different high voltage-activated Ca 2ϩ channels (A1S-, A1C-, A1D-, A1A-, A1B-, A1E subunits) has been investigated intensively during the last decade [3,4]. They are subdivided into two different families, based on their primary sequence. The A1S, A1C, and A1D subunits are sensitive against the classical blockers of L-type Ca 2ϩ channels, the dihydropyridines, the phenylalkylamines and the benzothiazepines. The subfamily of the non-L-type A1 subunits share, among each other, a functional interaction with inhibitory G proteins [5]. The Ntype Ca 2ϩ channels (A1B) are blocked by ω-conotoxin-GVIA, the P-type and Q-type Ca 2ϩ channels (A1A) are discriminated byCorrespondence to T. Schneider,