Rolf Vajna

New isoform of the neuronal Ca2+ channel α1E subunit in islets of Langerhans and kidney

The expression of Ca2+ channel alpha1E isoforms has been analyzed in different cell lines, embryoid bodies and tissues. The comparison of the different cloned alpha1E cDNA sequences led to the prediction of alpha1E splice variants. Transcripts of two cloned alpha1E isoforms, which are discriminated by a carboxy terminal 129-bp sequence, have been detected in different cell lines and tissues. Transcripts of the shorter alpha1E 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 isoform-specific antibodies. Transcripts of the longer alpha1E 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 betaTC-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 alpha1E in cell lines and tissues shows that the wide expression of alpha1E has to be specified by identifying the corresponding isoforms in each tissue. In islets of Langerhans and in kidney, a distinct isoform called alpha1Ee has been determined by RT-PCR, while in cerebellum a set of different alpha1E 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 alpha1Ee in islets of Langerhans and kidney leads to the suggestion that alpha1E might be involved in the modulation of the Ca2+-mediated hormone secretion.

Immunodetection of α1E Voltage-gated Ca2+ Channel in Chromogranin-positive Muscle Cells of Rat Heart, and in Distal Tubules of Human Kidney

The calcium channel α1E subunit was originally cloned from mammalian brain. A new splice variant was recently identified in rat islets of Langerhans and in human kidney by the polymerase chain reaction. The same isoform of α1E was detected in rat and guinea pig heart by amplifying indicative cDNA fragments and by immunostaining using peptide-specific antibodies. The apparent molecular size of cardiac α1E was determined by SDS-PAGE and immunoblotting (218 ± 6 kD; n = 3). Compared to α1E from stably transfected HEK-293 cells, this is smaller by 28 kD. The distribution of α1E in cardiac muscle cells of the conducting system and in the cardiomyoblast cell line H9c2 was compared to the distribution of chromogranin, a marker of neuroendocrine cells, and to the distribution of atrial natriuretic peptide (ANP). In serial sections from atrial and ventricular regions of rat heart, co-localization of α1E with ANP was detected in atrium and with chromogranin A/B in Purkinje fibers of the conducting system in both rat atrium and ventricle. The kidney is another organ in which natriuretic peptide hormones are secreted. The detection of α1E in the distal tubules of human kidney, where urodilatin is stored and secreted, led to the conclusion that the expression of α1E in rat heart and human kidney is linked to regions with endocrine functions and therefore is involved in the Ca2+-dependent secretion of peptide hormones such as ANP and urodilatin.

Immunohistochemical Detection of α1E Voltage-gated Ca2+ Channel Isoforms in Cerebellum, INS-1 Cells, and Neuroendocrine Cells of the Digestive System

Polyclonal antibodies were raised against a common and a specific epitope present only in longer α1E isoforms of voltage-gated Ca2+ channels, yielding an “anti-E-com” and an “anti-E-spec” serum, respectively. The specificity of both sera was established by immunocytochemistry and immunoblotting using stably transfected HEK-293 cells or membrane proteins derived from them. Cells from the insulinoma cell line INS-1, tissue sections from cerebellum, and representative regions of gastrointestinal tract were stained immunocytochemically. INS-1 cells expressed an α1E splice variant with a longer carboxy terminus, the so-called α1Ee isoform. Similarily, in rat cerebellum, which was used as a reference system, the anti-E-spec serum stained somata and dendrites of Purkinje cells. Only faint staining was seen throughout the cerebellar granule cell layer. After prolonged incubation times, neurons of the molecular layer were stained by anti-E-com, suggesting that a shorter α1E isoform is expressed at a lower protein density. In human gastrointestinal tract, endocrine cells of the antral mucosa (stomach), small and large intestine, and islets of Langerhans were stained by the anti-E-spec serum. In addition, staining by the anti-E-spec serum was observed in Paneth cells and in the smooth muscle cell layer of the lamina muscularis mucosae. We conclude that the longer α1Ee isoform is expressed in neuroendocrine cells of the digestive system and that, in pancreas, α1Ee expression is restricted to the neuroendocrine part, the islets of Langerhans. α1E therefore appears to be a common voltage-gated Ca2+ channel linked to neuroendocrine and related systems of the body. (J Histochem Cytochem 47:981–993, 1999)

Structural diversity of the voltage‐dependent Ca2+ channel α1E‐subunit

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 α1 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 α1E 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 α1E transcripts have been analysed by reverse transcription–polymerase chain reaction and by sequencing of amplified fragments. For the II–III loop three different α1E 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 α1E cDNA fragments of the carboxy terminus are identified in vivo. Two different α1E 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 α1E 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 α1E isoforms changes during development. In human cerebellum the longer α1E isoform is expressed predominantly. Although, it has not been possible to assign functional differences to the two α1E constructs tested in vitro, the expression pattern of the structurally related isoforms may have functional importance in vivo.

Embryonic stem-cell derived neurones express a maturation dependent pattern of voltage-gated calcium channels and calcium-binding proteins

There are remarkable changes of calcium binding proteins and voltage dependent Ca2+ channel subtypes during in vitro differentiation of embryonic stem cell derived neurons. To observe these maturation dependent changes neurones were studied using combined immunohistochemical, patch clamp and videomicroscopic time lapse techniques. Embryonic stem cell derived neuronal maturation proceeds from apolar to bi‐ and multipolar neurones, expressing all Ca2+ channel subtypes. There is, however, a clear shift in channel contribution to whole cell current from apolar neurones with mainly N‐ and L‐type channel contribution in favour of P/Q‐ and R‐type participation in bi‐ and multipolar cells. Expression of the calcium binding protein parvalbumin could be detected in bipolar, while calretinin and calbindin was preferentially found in multipolar neurones. Our data provides new insights into fundamental neurodevelopmental mechanisms related to Ca2+ homeostasis, and clarifies contradictory reports on the development of Ca2+ channel expression using primary cultures of neurones already committed to certain brain compartments.

Ca2+-sensitive regulation of E-type Ca2+ channel activity depends on an arginine-rich region in the cytosolic II–III loop

Ca2+‐dependent regulation of L‐type and P/Q‐type Ca2+ channel activity is an important mechanism to control Ca2+ entry into excitable cells. Here we addressed the question whether the activity of E‐type Ca2+ channels can also be controlled by Ca2+. Switching from Ba2+ to Ca2+ as charge carrier increased within 50 s, the density of currents observed in HEK‐293 cells expressing a human Cav2.3d subunit and slowed down the inactivation kinetics. Furthermore, with Ca2+ as permeant ion, recovery from inactivation was accelerated, compared to the recovery process recorded under conditions where the accumulation of [Ca2+]i was prevented. In a Ba2+ containing bath solution the Ca2+‐dependent changes of E‐type channel activity could be induced by dialysing the cells with 1 µm free [Ca2+]i suggesting that an elevation of [Ca2+]i is responsible for these effects. Deleting 19 amino acids in the intracellular II–III linker (exon 19) as part of an arginine‐rich region, severely impairs the Ca2+ responsiveness of the expressed channels. Interestingly, deletion of an adjacent homologue arginine‐rich region activates channel activity but now independently from [Ca2+]i. As a positive feedback‐regulation of channel activity this novel activation mechanism might determine specific biological functions of E‐type Ca2+ channels.

The cytosolic II–III loop of Cav2.3 provides an essential determinant for the phorbol ester‐mediated stimulation of E–type Ca2+ channel activity

There is growing evidence that E‐type voltage dependent Ca2+ channels (Cav2.3) are involved in triggering and controlling pivotal cellular processes like neurosecretion and long‐term potentiation. The mechanism underlying a novel Ca2+ dependent stimulation of E‐type Ca2+ channels was investigated in the context of the recent finding that influx of Ca2+ through other voltage dependent Ca2+ channels is necessary and sufficient to directly activate protein kinase C (PKC). With Ba2+ as charge carrier through Cav2.3 channel α1 subunits expressed in HEK‐293 cells, activation of PKC by low concentrations of phorbol ester augmented peak IBa by approximately 60%. In addition, the non‐inactivating fraction of IBa was increased by more than three‐fold and recovery from short‐term inactivation was accelerated. The effect of phorbol ester on IBa was inhibited by application of the specific PKC inhibitor bisindolylmaleimide I. With Ca2+ as charge carrier, application of phorbol ester did not change the activity of Cav2.3 currents but they were modified by the PKC inhibitor bisindolylmaleimide I. These results suggest that with Ca2+ as charge carrier the incoming Ca2+ can activate PKC, thereby augmenting Ca2+ influx into the cytosol. No modulation of Cav2.3 channels by PKC was observed when an arginine rich region in the II–III loop of Cav2.3 was eliminated. Receptor independent stimulation of PKC and its interaction with Cav2.3 channels therefore represents an important positive feedback mechanism to decode electrical signals into a variety of cellular functions.