Udo Klöckner

Cloning and Expression of a Novel Member of the Low Voltage-Activated T-Type Calcium Channel Family

Low voltage-activated Ca2+ 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, α1I or CavT.3, from rat brain. Northern analysis indicated that it is predominantly expressed in brain. Expression of the cloned channel in either Xenopusoocytes 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 α1G and α1H and to the high voltage-activated channels formed by α1Eβ3. The α1I channels opened after small depolarizations of the membrane similar to α1G and α1H but at more depolarized potentials. The kinetics of activation and inactivation were dramatically slower, which allows the channel to act as a Ca2+ 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 α1I 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 Ba2+ solutions. Based on its brain distribution and novel gating properties, we suggest that α1I plays important roles in determining the electroresponsiveness of neurons, and hence, may be a novel drug target.

Comparison of the Ca2 + currents induced by expression of three cloned α1 subunits, α1G, α1H and α1I, of low-voltage-activated T-type Ca2 + channels

Expression of rat α1G, human α1H and rat α1I subunits of voltage‐activated Ca2u200a+u200a channels in HEK‐293 cells yields robust Ca2u200a+u200a inward currents with 1.25u2003mm Ca2u200a+u200a as the charge carrier. Both similarities and marked differences are found between their biophysical properties. Currents induced by expression of α1G show the fastest activation and inactivation kinetics. The α1H and α1I 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 α1G and α1H deactivate with time constants of up to 6u2003ms at a test potential of −u200a80u2003mV, but currents induced by α1I deactivate about three‐fold faster. Recovery from short‐term inactivation is more than three‐fold slower for currents induced by α1H and α1I in comparison to α1G. In contrast to these characteristics, reactivation after long‐term inactivation was fastest for currents arising from expression of α1I and slowest in cells expressing α1H calcium channels. The calcium inward current induced by expression of α1I is increased by positive prepulses while currents induced by α1H and α1G show little (u200a<u200au20035%) or no facilitation. The data thus provide a characteristic fingerprint of each channels activity, which may allow correlation of the α1G, α1H and α1I induced currents with their in vivo counterparts.

Endothelin depolarizes myocytes from porcine coronary and human mesenteric arteries through a Ca-activated chloride current.

The effect of endothelin (ET) on membrane potential and current was studied in myocytes isolated from porcine coronary or from human mesenteric arteries at 3.6 mM extracellular Ca2+ concentration and 37° C. ET (1–100 nM) induced cell shortening and membrane depolarization from a resting potential of −50 mV to about −15 mV. Ca currents (ICa, L-type) were transiently reduced by ET. At −50 mV, ET induced an inward current that peaked within 2 s and fell within 10 s to a sustained level. The current could be enlarged by reducing bath extracellular Cl− ion concentration, but removal of extracellular Na+ ions had no effect. The voltage dependence suggests that the ET-induced current is a Cl current (ICl) at potentials negative to −30 mV; at more positive potentials K currents (IK, Ca) are superimposed. The effects of ET on ICa, ICl, IK, Ca contraction were prevented by intracellular Ca chelators, suggesting a Ca-dependent activation mechanism. The ET effects were abolished by pretreatment with 20 mM caffeine or prior cell-dialysis with heparin [thought to block inositol triphosphate-induced sarcoplasmic reticular Ca release]. The results suggest that ET releases Ca from the SR through a phosphoinositol response and that the released Ca acts as second messenger in modulating the membrane currents.

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.

Alternate splicing in the cytosolic II-III loop and the carboxy terminus of human E-type voltage-gated Ca^ channels : electrophysiological characterization of isoforms

There is growing evidence that Ca(v)2.3 (alpha1E, E-type) transcripts may encode the ion-conducting subunit of a subclass of R-type Ca(2+) channels, a heterogeneous group of channels by definition resistant to blockers of L-, N-, and P/Q-type Ca(2+) channels. To understand whether splice variation of Ca(v)2.3 contributes to the divergence of R-type channels, individual variants of Ca(v)2.3 were constructed and expressed in HEK-293 cells. With Ba(2+) as charge carrier, the tested biophysical properties were similar. In Ca(2+), the inactivation time course was slower and the recovery from short-term inactivation was faster; however, this occurred only in variants containing a 19-amino-acid-long insertion, which is typical for neuronal Ca(v)2.3 Ca(2+) channel subunits. This different Ca(2+) sensitivity is not responsible for the major differences between various R-type channels, and future studies might clarify its importance for in vivo synaptic or dendritic integration and the reasons for its loss in endocrine Ca(v)2.3 splice variants.

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.

Native-type DHP-sensitive calcium channel currents are produced by cloned rat aortic smooth muscle and cardiac α1 subunits expressed in Xenopus laevis ooeytes and are regulated by α2- and β-subunits

Native tissue‐like L‐type voltage‐dependent calcium channels (L‐VDCCs) were expressed by in vitro transcribed cRNA injection of rat aorta or rabbit cardiac α1 subunit into Xenopus laevis oocytes. Co‐injection of VSM‐α1 with the cloned skeletal muscle β‐subunit (SK‐β) of the L‐type VDCC significantly increased the expressed peak current amplitude without significant changes in kinetics. Similar results were obtained by co‐injection of cardiac α1 (DSHT‐α1) the cloned skeletal α2‐subunit (SK‐α2) or with SK‐β. The oocytes co‐expressing cRNAs retained L‐type VDCC pharmacology.

Isoforms of α1E voltage-gated calcium channels in rat cerebellar granule cells: Detection of major calcium channel α1-transcripts by reverse transcription–polymerase chain reaction

Abstract In primary cultures of rat cerebellar granule cells, transcripts of voltage-gated Ca 2+ channels have been amplified by reverse transcription–polymerase chain reaction and identified by sequencing of subcloned polymerase chain reaction products. In these neurons cultured for six to eight days in vitro , fragments of the three major transcripts α1C, α1A, and α1E are detected using degenerated oligonucleotide primer pairs under highly stringent conditions. Whole-cell Ca 2+ current recordings from six to eight days in vitro granule cells show that most of the current is due to L-type (25%), P-type (33%) and R-type (30%) Ca 2+ channels. These data support the correlation between α1A and P-type Ca 2+ channels (G1) and between α1E and R-type channels (G2 and G3). By including specific primer pairs for α1E the complimentary DNA fragments of indicative regions of α1E isoforms are amplified corresponding to the three most variable regions of α1E, the 5′-end, the II/III-loop, and the central part of the 3′-end. Although the complementary DNA fragments of the 5′-end of rat α1E yield a uniform reverse transcription–polymerase chain reaction product, its structure is unusual in the sense that it is longer than in the cloned rat α1E complementary DNA. It corresponds to the α1E isoform reported for mouse and human brain and is also expressed in cerebellum and cerebrum of rat brain as the major or maybe even the only variant of α1E. While fragments of a new rat α1E isoform are amplified from the 5′-end, three known fragments of the II/III-loop and two known isoforms homologue to the 3′-coding region are detected, which in the last case are discriminated by a 129xa0base pair insertion. The shift of the α1E expression from a pattern seen in cerebellum (α1Ee) to a pattern identified in other regions of the brain (α1E-3) is discussed. These data show that: (i) α1E is expressed in rat brain as a structural homologue to the mouse and human α1E; and (ii) rat cerebellar granule cells in primary culture express a set of α1E isoforms, containing two different sized carboxy termini. Since no new transcripts of high-voltage-activated Ca 2+ channels genes are identified using degenerate oligonucleotide primer pairs, the two isoforms differentiated by the 129xa0base pair insertion might correspond to the two R-type channels, G2 and G3, characterized in these neurons. Functional studies including recombinant cells with the different proposed isoforms should provide more evidence for this conclusion.

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 50u2003s, 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 1u2003µm 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 (exonu200319) 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.