alpha 1D (Cav1.3) Subunits Can Form L-type Ca2+ Channels Activating at Negative Voltages*

Alexandra KoschakDagger , Daniel ReimerDagger , Irene HuberDagger , Manfred GrabnerDagger , Hartmut GlossmannDagger , Jutta Engel§, and Jörg StriessnigDagger

From the Dagger  Institut für Biochemische Pharmakologie, Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria and the § Physiologisches Institut II/Sektion Sensorische Biophysik, HNO-Klinik, Universität Tübingen, Röntgenweg 11, D-72076 Tübingen, Germany

Received for publication, February 15, 2001, and in revised form, March 29, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In cochlea inner hair cells (IHCs), L-type Ca2+ channels (LTCCs) formed by alpha 1D subunits (D-LTCCs) possess biophysical and pharmacological properties distinct from those of alpha 1C containing C-LTCCs. We investigated to which extent these differences are determined by alpha 1D itself by analyzing the biophysical and pharmacological properties of cloned human alpha 1D splice variants in tsA-201 cells. Variant alpha 1D8A, containing exon 8A sequence in repeat I, yielded alpha 1D protein and L-type currents, whereas no intact protein and currents were observed after expression with exon 8B. In whole cell patch-clamp recordings (charge carrier 15-20 mM Ba2+), alpha 1D8A - mediated currents activated at more negative voltages (activation threshold, -45.7 versus -31.5 mV, p < 0.05) and more rapidly (tau act for maximal inward currents 0.8 versus 2.3 ms; p < 0.05) than currents mediated by rabbit alpha 1C. Inactivation during depolarizing pulses was slower than for alpha 1C (current inactivation after 5-s depolarizations by 90 versus 99%, p < 0.05) but faster than for LTCCs in IHCs. The sensitivity for the dihydropyridine (DHP) L-type channel blocker isradipine was 8.5-fold lower than for alpha 1C. Radioligand binding experiments revealed that this was not due to a lower affinity for the DHP binding pocket, suggesting that differences in the voltage-dependence of DHP block account for decreased sensitivity of D-LTCCs. Our experiments show that alpha 1D8A subunits can form slowly inactivating LTCCs activating at more negative voltages than alpha 1C. These properties should allow D-LTCCs to control physiological processes, such as diastolic depolarization in sinoatrial node cells, neurotransmitter release in IHCs and neuronal excitability.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
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L-type Ca2+ channels (LTCCs)1 form a family of voltage-gated Ca2+ channels with high sensitivity to dihydropyridine (DHP) Ca2+ channel modulators. Their Ca2+-selective pore is formed by different DHP-sensitive alpha 1 subunit isoforms (alpha 1S, alpha 1C, alpha 1D, alpha 1F) together with auxiliary subunits, including alpha 2-delta and beta  subunits (1-3). Whereas alpha 1S and alpha 1F expression is restricted to skeletal muscle and the retina, respectively, LTCCs formed by alpha 1C (C-LTCCs) and alpha 1D (D-LTCCs) subunits are widely expressed in neuronal and (neuro)endocrine cells as well as in electrically excitable cells in the cardiovascular system (4-9). In most cases, both channel types are even found in the same cells, with D-LTCCs usually being the much less abundant isoform (7).

Using D-LTCC-deficient mice, we have previously demonstrated that inward currents through alpha 1D form LTCCs with biophysical and pharmacological properties distinct from C-LTCCs (4, 6). These include a more negative range of current activation and slower current inactivation during depolarizations, allowing these channels to mediate long lasting Ca2+ influx during weak depolarizations. Such properties allow LTCCs to control tonic neurotransmitter release in hair cells (5, 10), diastolic depolarization in the sinoatrial node (11), and electrical excitability of neurons (12-15).

In addition to these different biophysical properties, it has also been postulated that D-LTCCs may exhibit a lower sensitivity to DHP channel blockers (4, 16). However, this has never been proven or quantified in a comparative study of the DHP sensitivity of D- and C-LTCCs. At present it is also unclear to which extent these biophysical and pharmacological differences are determined by the alpha 1D subunit itself, by known accessory Ca2+ channel subunits, or by other Ca2+ channel-associated proteins.

By analyzing the biophysical and pharmacological properties of cloned human alpha 1D splice variants in tsA-201 cells, we provide evidence that most of the biophysical differences described above are determined by alpha 1D. We also demonstrate that alternative splicing of exon 8 is critically affecting the expression of functional alpha 1D protein in tsA-201 cells. Our analysis also revealed that, in functional experiments, D-LTCCs display lower sensitivity for DHPs than alpha 1C, despite similar affinity for the DHP binding pocket.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cloning of Human alpha 1D (CaV1.3) Subunits-- alpha 1D cDNAs were obtained by reverse transcription-polymerase chain reaction from human pancreas poly(A)+ RNA (CLONTECH) using proofreading Pfu TurboTM DNA polymerase (Stratagene). First strand cDNA was synthesized using 1 µg of poly(A)+ RNA employing Ready-To-GoTM T-primed first-strand reaction kit (Amersham Pharmacia Biotech). Five alpha 1D fragments with different native or artificial restriction sites were amplified from the first strand cDNA using suitable primer pairs. Fragments were as follows (nucleotide numbering according to Ref. 9; asterisks indicate artificial restriction sites introduced by polymerase chain reaction): F1, SalI*-SpeI (nucleotides 1-758), F2, SpeI-SphI (nucleotides 758-1405), F3/4 ClaI-HindIII (nucleotides 1353-3993), F5 HindIII-XhoI* (nucleotides 3993-5340), and F6 XhoI*-BamHI* (nucleotides 5340-6498). Fragments were subcloned into the expression vectors pBluescript SK+® (Stratagene) or pSport-1® (Life Technologies, Inc.). Two F2 fragments containing different splice variants (8A and 8B; Ref. 9) were obtained and used to construct alpha 1D8A and alpha 1D8B. The sequence integrity of all fragments was determined by DNA sequencing. The complete alpha 1D cDNA was constructed by generating subclone F5/6 by coligating the HindIII-XhoI* fragment (F5) and the XhoI*-BamHI* fragment (F6) into pBluescript SK+. F1 and F2 fragments were coligated into the corresponding SalI/SphI RE sites of the plasmid containing fragment F3/4. The resulting SalI*-HindIII fragment (F1-4) was ligated into the SalI/HindIII restriction sites of subclone F5/6. For heterologous expression studies, the alpha 1D cDNA was cloned into the mammalian expression plasmids pGFP- and pGFP+ (17). Cloning into pGFP+ generates a fusion protein of alpha 1D with N-terminally located green fluorescent protein (GFP).

Transient Expression of alpha 1D Splice Variants in tsA-201 Cells-- tsA-201 cells were maintained in Dulbecco's modified Eagle's medium/Coon's F-12 medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal calf serum (Sebak), 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin at 37 °C and 7% CO2. For transient Ca2+ channel expression, cells were grown to 80% confluence and plated at dilutions of 1:10 on glass coverslips 12 h before transfection by lipofection (using N-[1-(2,3-dioleoy(oxy)propyl]N,N,N-trimethylammoniummeshyl-sulfate, or Fugene-6; Roche Molecular Biochemicals) or Ca2+ phosphate precipitation using standard protocols. Human alpha 1D, rat alpha 1D (cloned into expression plasmid pCMV6b; Ref. 18), or rabbit alpha 1C-a (19) subunits were expressed together with alpha 2delta (20), bovine (21), or rat beta 2a (22) or rat beta 3 subunits (23). The cells were incubated at 30 °C and 5% CO2 12 h after transfection for 2-5 days prior to recording or membrane preparation.

Membrane Preparation and (+)-[3H]Isradipine Binding-- Membranes from tsA-201 cells transfected with 4.5 µg of alpha 1, 3.5 µg of alpha 2-delta , 2.5 µg of beta 1a or beta 3 subunit, and 4.5 µg of pUC18 carrier DNA in 10-cm culture dishes were prepared as described (24). Binding experiments with (+)-[3H]isradipine were performed in a final assay volumes of 0.5 or 1 ml (24). Experimental details are given in the legend to Fig. 5. Kinetic experiments were performed in the absence of added Ca2+. Nonspecific binding was determined in the presence of 1 µM unlabeled isradipine. Serial dilutions of drugs were made in Me2SO (final Me2SO concentration < 1% (v/v)).

Electrophysiological Recordings-- Whole cell patch clamp experiments were carried out at room temperature using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Currents were recorded at sampling rates of 5 or 25 kHz, low pass-filtered at 2 or 5 kHz, and recorded directly onto a personal computer equipped with pCLAMP version 7.0. Borosilicate glass pipettes had a resistance of 2-4 megohms when filled with internal solution. Capacitance and series resistance compensations of 60-80% were used. The voltage error due to uncompensated series resistance was 1.2 ± 0.6 mV. The average membrane time constant was 83 ± 6 µs. Unless stated otherwise, Ba2+ currents (IBa) through LTCCs were measured with the following solutions: Pipette solution (in mM): 135 mM CsCl, 10 mM Cs-EGTA, and 1 mM MgCl2, adjusted to pH 7.4 with CsOH; bath solution (in mM): 15-20 mM BaCl2, 10 HEPES, 150 mM choline Cl, and 1 mM MgCl2, adjusted to pH 7.4 with CsOH. Run-down of IBa was typically less than 10% in 3 min. All voltages were corrected for a liquid junction potential of -10 mV. Leak and capacitative currents were measured using hyperpolarizing pulses. Raw currents were corrected for linear leak currents. The voltage-dependence of activation was determined from IV curves obtained by step depolarizations from a holding potential of -90 mV to various test potentials. IV curves were fitted according to Equation 1.


I=G<SUB><UP>max</UP></SUB>(V−V<SUB><UP>rev</UP></SUB>)/{1+<UP>exp</UP>[(V<SUB>0.5,<UP>act</UP></SUB>−V)/k]} (Eq. 1)
Vrev is the extrapolated reversal potential of IBa, V is the membrane potential, I is the peak current, Gmax is the maximum conductance of the cell, V0.5,act is the voltage for half-maximal activation, and kact is the slope factor of the Boltzmann term. The time course of current activation was fitted using an exponential function.
I(t)=A<SUB>0</SUB>×<UP>exp</UP>(<UP>−</UP>t/&tgr;<SUB>0</SUB>)+C (Eq. 2)
I(t) is the current at time t after the depolarization, A0 is the steady state current amplitude with the respective time constant of activation, tau 0, and C represents the remaining steady state current. The rate of inactivation was assessed by both the percentage of current that had inactivated after 2 s, and by fitting the current traces to the biexponential function, yielding time constants for a fast (tau fast) and a slow (tau slow) component.
I(t)=A<SUB><UP>fast</UP></SUB>[<UP>exp</UP>(<UP>−</UP>t/&tgr;<SUB><UP>fast</UP></SUB>)]+A<SUB><UP>slow</UP></SUB>[<UP>exp</UP>(<UP>−</UP>t/&tgr;<SUB><UP>slow</UP></SUB>)]+C (Eq. 3)
Voltage dependence of inactivation under quasi-steady state conditions was measured using a multi step protocol to account for run-down. A control test pulse (60 ms to the voltage of peak current, Vmax) was followed by a 2-s step to -90 mV, followed by a 5-s conditioning step, and a subsequent test pulse to Vmax. The start-to-start interval was 20 s at a holding potential of -90 mV. Inactivation during the 5-s conditioning pulse was calculated as follows.
% <UP>inactivation</UP>=(1−(I<SUB><UP>Ba,test</UP></SUB><UP>/</UP>I<SUB><UP>Ba,control</UP></SUB>))×100 (Eq. 4)
IBa,control and IBa,test are the current amplitudes at Vmax before and after the 5-s conditioning pulse, respectively.

Inactivation curves were fitted to a Boltzmann equation as described (25). Effects of DHPs were monitored continuously using 0.1-Hz depolarizing pulses to Vmax. DHPs were dissolved in the external recording solution from a 10 mM stock solution in dimethyl sulfoxide and perfused through a microcapillary onto cells using a gravity-driven perfusion system. Only cells exhibiting stable currents (run down <15% during the first 60 s) were used for analysis of DHP effects. The DHPs isradipine and BayK8644 were employed as their racemic mixtures.

Transfected cells were visualized as cotransfected GFP fluorescence Ca2+ currents in IHCs were measured as previously described in cochleae isolated from 2-4-day-old animals (4).

Immunoblotting and preparation of affinity-purified sequence-directed antibodies was carried out as described (4, 26) using antibodies described in the legend to Fig. 1.

Statistics-- All data are presented as mean ± S.E. for the indicated number of experiments. Statistical significance was determined by unpaired Student's t test. Data were analyzed using Clampfit (Axon Instruments) and Origin® 5.0 (Microcal).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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We cloned two different cDNAs encoding full-length alpha 1D subunits (Fig. 1A) from human pancreatic tissue, containing alternatively spliced exons 8A or 8B (alpha 1D8A, alpha 1D8B), which results in a six-amino acid difference in the pore region of repeat I. Their sequence is identical to a previously cloned cDNA (9) but both splice variants lack exons 32 and 44. Heterologous expression in tsA-201 cells together with auxiliary subunits yielded DHP-sensitive L-type currents for alpha 1D8A (100 of 154 patched GFP-expressing cells gave measurable IBa) but not for alpha 1D8B. Similarly, no current was measured under our experimental conditions after transfection with rat alpha 1D cDNA (18), which also contains exon 8B. To determine if the absence of Ca2+ current resulted in the expression of a non-functional subunit or was due to the absence of alpha 1D protein, alpha 1D subunit expression was quantified by immunoblot analysis of transfected tsA-201 cell membranes (Fig. 1B). A full-length form of alpha 1D protein was only detected for alpha 1D8A but not for rat and human alpha 1D8B. After transfection of cells with alpha 1D8B, alpha 1D immunoreactivity was only associated with polypeptides smaller than the expected full-length form (Fig. 1), suggesting that alpha 1D8B protein underlies proteolytic degradation in tsA-201 cells. As a cloning artifact was ruled out by DNA sequencing (see "Experimental Procedures"), our data suggest that the six-amino acid residue difference prevents effective alpha 1D Ca2+ subunit expression in tsA-201 cells.


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Fig. 1.   A, position of alternatively spliced exons 8, 32, 44 in alpha 1D subunits. The expressed alpha 1D cDNAs (alpha 1D8A and alpha 1D8B) are identical to the cDNA reported by Williams et al. (9) but lack exons 32 and 44 (position indicated by arrows). They contain exon 8A or 8B in repeat I (bold line and black box). A sequence alignment of the predicted amino acid sequences encoded by exon 8A and exon 8B is also shown. B, expression of alpha 1D subunits in tsA-201 cells. Heterologous expression of human alpha 1D8A, alpha 1D8B, and rat alpha 1D8B in tsA-201 cells is shown. Cells were transfected with alpha 1 together with alpha 2delta and beta  subunit DNA as described under "Experimental Procedures." alpha 1D subunit expression was analyzed in immunoblots of membranes prepared from lysed cells after separation on 8% SDS-polyacrylamide gels using sequence-directed antibodies raised against alpha 1D residues 30-48 (anti-alpha 1D30-48) and 2121-2137 (anti-alpha 1D2121-2137) (9) or an antibody recognizing all alpha 1 subunits (alpha 1com, raised against residues 1382-1400 of alpha 1S). Arrows indicate specific alpha 1D immunoreactivity absent in mock transfected cells. One representative experiment (of n > 2) is shown.

Next we tested if alpha 1D8A could give rise to L-type currents with the characteristics described recently in chick and mouse cochlea (4, 5, 16). These include low activation threshold, fast activation kinetics, slow inactivation, and lower apparent DHP antagonist sensitivity. After transfection using Ca2+ phosphate precipitation, the alpha 1D current density was similar to alpha 1C-a mediated currents (Table I). With 15 mM Ba2+ as the charge carrier, the threshold of activation for alpha 1D8A was found at about 15 mV more hyperpolarized potentials (-45.7 ± 0.5 mV, n = 38) as compared with alpha 1C-a (-31.5 ± 0.5 mV, n = 16) (Table I, Fig. 2A). The maximum of the current-voltage relationship (Vmax) as well as the potential of half-maximal IBa activation (V0.5,act) was also shifted significantly to hyperpolarized potentials without affecting the slope of the activation curve (Table I). Depolarizations to Vmax revealed that IBa through alpha 1D8A showed a monoexponential activation time course about 3-fold faster than for alpha 1C-mediated currents (Table I, Fig. 2B). As for alpha 1C-a, the speed of alpha 1D8A activation increased at more positive voltages. More rapid activation of alpha 1D8A was consistently found over a voltage range from -30 to +30 mV (Fig. 2C).

                              
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Table I
Biophysical properties of D- and C-LTCCs expressed in tsA-201 cells
The half-maximal voltage for activation (V0.5,act), the slope for activation (kact), the maximum of the current-voltage relationship (Vmax), and the slope for inactivation (kinact) were obtained by fitting the data as described under "Experimental Procedures." The activation threshold is defined in the legend to Fig. 2. The time course of current activation was fitted using a single exponential function. tau act is given for IBa elicited by test pulses to Vmax. Data are given as mean ± S.E. *, statistically significant difference (p < 0.05).


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Fig. 2.   alpha 1D8A and alpha 1C-a show distinct activation properties. Current recordings were carried out as described under "Experimental Procedures" with 20 mM Ba2+ as charge carrier. Representative experiments are shown in A and B. For statistics see Table I. A, normalized IV curves for alpha 1D8A and alpha 1C-a. The activation threshold, determined as the test potential at which 5% of maximal IBa was activated, was -45.7 ± 0.5 mV (filled circles) and -31.5 ± 0.5 mV (open circles) for alpha 1D8A and alpha 1C, respectively. B, the kinetics of current activation was determined by depolarizing pulses to Vmax. Representative traces for alpha 1D8A and alpha 1C-a show monoexponential activation time courses with time constants (tau act) of 0.41 and 1.45 ms, respectively. C, voltage dependence of tau act for alpha 1D8A (filled circles) and alpha 1C-a (open circles). tau act was determined for currents elicited by test pulses to the indicated potentials. *, statistically significant difference (p < 0.05).

To test if alpha 1D8A also mediates the slowly inactivating L-type currents observed in IHCs, we compared current inactivation during long test pulses with the inactivation time course of (largely alpha 1D-mediated; Ref. 4) L-type currents in IHCs and heterologously expressed alpha 1C. During a 5-s depolarizing test pulse from a holding potential of -90 mV to Vmax, 90 ± 2.2% (n = 6) of alpha 1D8A current inactivated (Fig. 3A). In contrast, only 9.3 ± 4.6% (n = 6) of IBa inactivated during a 5-s depolarizing pulse in IHCs. Although inactivation of alpha 1D8A current was faster than in IHCs, it was significantly slower than for alpha 1C-a (99 ± 1.3%, n = 4, p < 0.05; Fig. 3, A and B). Fig. 3A shows normalized currents of alpha 1C-a and alpha 1D8A, which both exhibit biexponential inactivation. Slower inactivation of alpha 1D8A was due to a 1.5-fold increase of the time constant for the slowly inactivating component (Fig. 3B) (alpha 1D8A: tau slow = 1.7 ± 0.2 s, n = 6; alpha 1C-a: tau slow = 1.1 ± 0.2 s, n = 4) and a decrease in the relative contribution of the fast component (alpha 1D8A: 45 ± 3%, tau fast = 0.187 ± 0.015 s; alpha 1C: 60 ± 6%, tau fast = 0.145 ± 0.022 s; n = 4-6).


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Fig. 3.   Inactivation properties of the L-type Ca2+ channel alpha 1D8A. An IBa was elicited by 5 s depolarizing pulses from a holding potential of -90 mV to Vmax. Representative traces of alpha 1D8A and alpha 1C-a (coexpressed with beta 3 and alpha 2delta ) were fit to a biexponential decay yielding the following time constants for the fast (tau fast) and the slow (tau slow) component (in seconds); the contribution of the fast and slow components to total IBa are given in parentheses: alpha 1D8A, 0.18 (40%) and 1.7 (48%); alpha 1C, 0.16 (69%) and 1.05 (30%). *, statistically significant difference (p < 0.05). B, inactivation time constants for the fast (tau fast) and the slow (tau slow) component (in seconds). Data are means ± S.E. for n = 4-6. *, statistically significant difference (p < 0.05). C, effects of beta -subunit expression on the inactivation properties of GFP-alpha 1D8A. Current inactivation was measured during 2-s depolarizing pulses to Vmax for rat beta 3, rat beta 2a, and bovine beta 2a: 82.8 ± 6.5% (n = 4); 34.5 ± 3.2%, n = 8; 38.1 ± 10.2%, n = 4). Slowing of inactivation was significant (p < 0.05; asterisk) only for rat beta 2a. D, voltage dependence of Ca2+ channel inactivation during 5-s depolarizing pulses. Inactivation was determined as described under "Experimental Procedures." Half-maximal inactivation potential (V0.5,inact) and the corresponding slope parameters (kinact) of alpha 1D8A (filled circles) and alpha 1C-a (open circles) are given in Table I.

beta 2a subunits stabilize slow inactivation of Ca2+ channel alpha 1 subunits (21, 27). We therefore investigated if the slower inactivation time course of alpha 1D currents of IHCs can be obtained by coexpression with rat or bovine beta 2a subunits (together with alpha 2delta subunits). As shown in Fig. 3C, current inactivation during 2-s depolarizing test pulses was slower upon coexpression of rat or bovine beta 2a than upon coexpression with beta 3 but could not account for the slow inactivation of alpha 1D in IHCs.

Steady-state inactivation of IBa was compared after coexpression of beta 3 subunits with 15 mM Ba2+ as the charge carrier. The midpoint voltage of the steady-state inactivation curve for alpha 1D8A was about 10 mV more negative (-42.7 ± 1.6 mV, n = 6) compared with alpha 1C-a (-27.6 ± 2.7 mV, n = 4) (Fig. 3D). Note that inactivation of alpha 1D was not complete during the 5-s conditioning prepulses.

The more negative activation threshold (alpha 1D8A: -37.2 ± 0.98, n = 13; alpha 1C-a: -27.6 ± 1.3, n = 16) was also observed when 15 mM Ca2+ were used as the charge carrier. alpha 1D8A showed pronounced Ca2+-induced inactivation kinetics. During test pulses to Vmax, about 70% of ICa inactivated within less than 400 ms (n = 6). Inactivation of alpha 1D8A current was slower and less complete than alpha 1C-a (data not shown).

Previous experiments suggested that the DHP antagonist sensitivity may be lower for D- than for C-LTCCs. However, this has never been proven by a systematic comparison of DHP antagonist effects on alpha 1D and alpha 1C-a currents under identical experimental conditions. We therefore measured the inhibition of both channel types by the DHP antagonist isradipine at various concentrations and at different holding potentials. At -90 mV holding potential, 300 nM isradipine completely inhibited alpha 1C-a current (98.3 ± 1.7%, n = 3) but only 30-40% of alpha 1D8A (Fig. 4). The concentration dependence of block revealed that isradipine sensitivity for alpha 1D8A was 8.5-fold lower (Fig. 4B) at this holding potential. Isradipine sensitivity increased by an order of magnitude at a more positive holding potential (-50 mV, Fig. 4B), demonstrating a voltage-dependent mechanism of DHP block as described previously for alpha 1C-a currents (28, 29).


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Fig. 4.   DHP sensitivity of alpha 1D8A and alpha 1C-a. For all experiments, alpha 1 subunits were coexpressed with beta 3 and alpha 2delta in tsA-201 cells. Charge carrier was 15 mM Ba2+. A, voltage dependence of IBa inhibition of alpha 1D8A by isradipine. IBa was elicited from a holding potential of -90 mV (filled circles) or -50 mV (open circles) before and during superfusion of the cell with isradipine-containing solution. Maximal IBa is plotted against time. The inset shows representative current traces (holding potential -50 mV) in the absence (control, at 40 s) and presence of 300 nM isradipine (at 120 s). B, the concentration-response relationship of peak IBa inhibition of alpha 1D8A (circles) and alpha 1C-a (triangles) by isradipine was determined at holding potentials of -90 mV (filled symbols) and -50 mV (open symbols; alpha 1D8A only). C, normalized IV curves for alpha 1D8A in the absence (control, open squares) and presence of the Ca2+ channel activator BayK8644 (5 µM, filled squares). D, corresponding peak current traces elicited by 30-ms depolarizations to Vmax in the absence and presence of BayK8644.

To determine if this sensitivity difference was due to a lower affinity of isradipine for alpha 1D8A, its affinity for the DHP binding pocket was determined in membranes prepared from transfected cells in radioligand binding experiments with (+)-[3H]isradipine (Fig. 5). Under identical experimental conditions, similar affinities were measured for alpha 1D8A (KD = 0.42 ± 0.06 nM, n = 10) and alpha 1C-a (KD = 0.68 ± 0.12 nM, n = 8) (Fig. 5A). Therefore, different affinities for the DHP drug binding pocket cannot account for the lower sensitivity observed in our functional studies. Dissociation of bound (+)-[3H]isradipine occurred about 8-fold faster from alpha 1D8A (k-1 = 32.6 ± 4.8 × 10-3 min-1, n = 3) than from alpha 1C-a (k-1 = 4.2 ± 0.1 × 10-3 min-1, n = 2) (Fig. 5B). This suggests that, despite similar binding affinity, binding kinetics differ between the two alpha 1 subunit isoforms.


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Fig. 5.   Reversible binding of (+)-[3H]isradipine to alpha 1D and alpha 1C-a expressed in tsA-201 cells. A, equilibrium saturation studies. Increasing concentrations of (+)-[3H]isradipine were incubated (37 °C, 120 min) with membrane protein (alpha 1D, 33 µg/ml; alpha 1C, 23 µg/ml) prepared from tsA-201 cells transfected with alpha 1D or alpha 1C-a together with alpha 2-delta  + beta  DNAs. Nonspecific binding was determined in the presence of 1 µM isradipine. Specific binding data were fitted to a monophasic saturation isotherm yielding the following binding parameters: alpha 1D8A, KD = 334 pM and Bmax = 1.72 pmol/mg; alpha 1C, KD = 503 pM and Bmax = 1.11 pmol/mg. B, dissociation experiments. Membrane protein from cells expressing alpha 1D (25 µg/ml) or alpha 1C-a (25 µg/ml) were incubated with 0.19 nM (+)-[3H]isradipine until equilibrium (B0) was reached. Dissociation was induced by rapid addition of a final concentration of 1 µM unlabeled isradipine. A semilogarithmic representation of the time-dependent decay of specific (+)-[3H]isradipine binding (B) is illustrated. Linear regression analysis (r > 0.98) yielded the following k-1 values: for alpha 1D8A, 27.8 × 10-3 × min-1; for alpha 1C, 4.3 × 10-3 × min-1).

alpha 1D8A Ca2+ channels coexpressed with beta 3 and alpha 2delta in tsA-201 cells also exhibited the typical current modulation by the Ca2+ channel activator BayK8644 (Fig. 4, C and D) similar to its actions in IHCs (4). BayK8644 increased the maximal IBa, produced a slight hyperpolarizing shift in the IV relationship and slowed current deactivation of the tail current induced by repolarization of the cell to the holding potential (Fig. 4D). The extent of maximal IBa stimulation was more pronounced in tsA-201 cells as compared with IHCs (7.5 ± 1.8 -fold, n = 6), versus 2.9-fold; Ref. 4).

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Here we present the first detailed patch clamp analysis of the biophysical properties of whole cell currents through Ca2+ channels formed by alpha 1D subunits after heterologous expression in mammalian cells. Our studies revealed that these subunits can form L-type Ca2+ channels with properties similar to D-LTCCs previously described in IHCs (4). We clearly demonstrate lower DHP antagonist sensitivity, more rapid activation kinetics, and slower inactivation for the alpha 1D8A splice variant as compared with alpha 1C-a currents. Another novel finding was that the lower DHP antagonist sensitivity is not due to lower binding affinity for the DHP binding pocket. Instead, it is only evident in the presence of membrane potential and therefore must involve a voltage-dependent mechanism affecting DHP antagonist sensitivity in functional studies. We were also able to demonstrate that the heterologous expression of alpha 1D in mammalian cells is critically affected by six amino acids in the pore region of repeat I.

Our previous experiments with mice lacking class D Ca2+ channels clearly revealed that alpha 1D subunits are required to form low voltage-activated LTCCs with the above described properties in mouse cochlear IHCs. However, from these experiments it remained unclear if all of these properties are inherent to alpha 1D or require the presence of a yet unidentified Ca2+ channel subunit.

Heterologous expression of alpha 1D8A together with alpha 2delta and beta 3 (or beta 2a) resulted in IBa with an activation threshold below -40 mV (15-20 mM Ba2+ as charge carrier) and thus are very similar to the activation range observed in the presence of 10 mM Ba2+ in IHCs (-40 to -50 mV, Ref. 4). The activation threshold for alpha 1D8A-mediated currents can be estimated to be about -60 mV at physiological Ca2+ concentrations (4). The activation time constants were significantly faster than for alpha 1C-a at most voltages and at positive voltages (20-40 mV : tau act = 0.44-0.56 ms; Fig. 2C) close to the value obtained in IHCs (0.31 ms; Ref. 4). Very similar values were also measured for V0.5,act (alpha 1D8A: -17.5 mV; IHC: -16.5 mV) and Vmax (alpha 1D8A: -5.5 mV; IHC: -2.1 mV; Ref. 4). Interestingly, alternative splicing has been observed in transmembrane segment S3 and the adjacent S3-S4 linker in repeat IV. This region affects the voltage-dependence of activation in voltage-gated Ca2+ (30), Na+ (31), and K+ (32) channels. It is therefore possible that alternative splicing in this region also affects the activation threshold of alpha 1D. In contrast to our constructs, a previously cloned human alpha 1D (9) contains exon 32 (and exon 44). Unfortunately, its biophysical and pharmacological properties have not been directly compared with alpha 1C under identical experimental conditions. Stable expression of this cDNA in HEK-293 cells (together with beta 3 and alpha 2delta , 20 mM Ba2+ as charge carrier) yielded channel currents with V0.5,act of +1.8 mV and a Vmax of about +20 mV (33). However, correction for junction potential would shift these values to more negative potentials by about 23 mV, resulting in activation parameters nearly identical to our alpha 1D8A subunit. Minor effects of exon 32 on channel gating cannot be ruled out and will require a more detailed analysis.

The only property not mimicked by our splice variant was the very slow inactivation of IHC currents during a depolarizing pulse. alpha 1D8A currents inactivate slower than alpha 1C-a but faster than IBa in IHCs. As compared with alpha 1C-a, the slower inactivation of alpha 1D8A can be explained by an increase of the contribution and the time constant of a slow component of IBa inactivation. It remains to be determined if alternative splicing or the presence of another auxiliary subunit is responsible for the slow inactivation of class D currents in IHCs.

The relatively weak channel block of IBa through class D LTCCs by DHP antagonists was now directly confirmed in our experiments. We describe for the first time that DHP inhibition of alpha 1D is voltage-dependent and favored at more positive voltages. As for alpha 1C-a, this indicates higher affinity for inactivated channels (28, 29) or induction of inactivated channel states (25). alpha 1D8A showed an approximately 10-fold lower sensitivity for block by the DHP antagonist isradipine than alpha 1C-a. This finding nicely explains the absence of major side effects expected from block of class D channels in humans at therapeutic plasma concentrations. This includes the lack of bradycardic actions expected from the block of class D channels in sinoatrial node cells as well as the absence of hearing disturbances resulting from the block of this channel type in cochlear IHCs (4). Instead, DHPs preferentially block class C LTCCs in the cardiovascular system (34). The higher DHP sensitivity of the smooth muscle alpha 1C splice variant (alpha 1C-b) as compared to the cardiac splice variant (alpha 1C-a; Ref. 28) in part explains the selectivity of DHPs for arterial smooth muscle, resulting in therapeutic antihypertensive effects in the absence of cardiodepression (34). The higher sensitivity of alpha 1C-b is also not due to a higher affinity of DHP antagonists for its DHP binding pocket (28). Instead, it could be explained by differences in the voltage dependence of alpha 1C block and is due to amino acid divergence between the two splice variants in transmembrane segment IS6. We propose that a similar mechanism must explain the lower DHP antagonist sensitivity of alpha 1D8A as compared with alpha 1C-a because the affinity for the DHP binding pocket is the same in these subunits.

Although we and others (9) found evidence for alternative splicing in segment IS6 (exons 8A and 8B, respectively), the role of exon 8B for alpha 1D function and DHP modulation could not be assessed. This was due to the absence of intact alpha 1D protein and currents after expression of our human alpha 1D construct in which exon 8A sequence was exchanged for exon 8B (alpha 1D8B). The inhibitory role of exon 8B on alpha 1D expression is further supported by our inability to transiently express full-length rat alpha 1D, which also contains exon 8B, despite the abundant presence of mRNA in the transfected cells.2 In accordance with our interpretation, the functional expression of exon 8B containing alpha 1D cDNAs isolated from rat brain (35) and chicken cochlea (36) has not yet been reported. Further studies must determine which of the 6 amino acids differing between these alternative exons account for this effect. It is possible that alternative splicing in IS6 serves as a molecular switch to modulate alpha 1D subunit expression on the post-transcriptional level.

Our experiments provide convincing evidence that alpha 1D8A-mediated L-type currents activate at slightly more positive voltages than recombinant T-type channels (37) but at more negative voltages than C-LTCCs as reported here. In this respect they resemble alpha 1E-mediated currents (37), which were originally classified as low voltage-activated (38). Although we cannot exclude that alternative splicing or biochemical modulation (e.g. phosphorylation) also allows other L-type alpha 1 subunit isoforms (including alpha 1C) to activate at such "intermediate" voltages, our data provide direct evidence that D-LTCCs can account for the "low voltage-activated" L-type currents described previously in neurons (15). We have developed mutant mice in which the DHP sensitivity of alpha 1C-subunits is dramatically reduced (39). Such animal models will allow direct determination of the role of D-LTCCs for L-type current components in various tissues.

Taken together, the evidence demonstrates that expression of alpha 1D subunits should enable cells to slowly inactivating voltage-gated Ca2+ influx in response to rather weak depolarizations. This property allows them to participate in important physiological functions such as tonic neurotransmitter release in IHCs (10) and control of diastolic depolarization in sinoatrial node (4). These properties also make them ideally suited to contribute to subthreshold Ca2+ signaling (e.g. in hippocampal pyramidal cells; Ref. 14) and to the plateau potential underlying bistable membrane behavior (e.g. in motoneurons; Refs. 12 and 13).

    ACKNOWLEDGEMENTS

We thank Dr. S. Seino for providing rat alpha 1D and beta 3 and Dr. A. Cahill for bovine beta 2a cDNA. We thank Drs. J. Platzer, S. Berjukov, and S. Hering for advice and E. Wappl for helpful comments on the manuscript.

    Note Added in Proof

In a report, which will be published later this year (Scholze et al., Mol. Endocrinol., in press), similar biophysical properties are reported for a Cav1.3 splice variant cloned from HIT-T15 cells after expression in Xenopus laevis oocytes.

    FOOTNOTES

* This work was supported by Austrian Science Fund Grants P12641 (to J. S.) and P-12689 (to H. G.), European Community Grant HPRN-CT2000-00082 (to J. S.), a grant from the Österreichische Nationalbank (to J. S.), and a grant from the Dr. Legerlotz Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. E-mail: joerg.striessnig@vibk.ac.at.

Published, JBC Papers in Press, April 2, 2001, DOI 10.1074/jbc.M101469200

1 The following abbreviations were used: LTCC, L-type Ca2+ channel; C-LTCC, L-type Ca2+ channel formed by alpha 1C; D-LTCC, L-type Ca2+ channel formed by alpha 1D; DHP, dihyropyridine; IBa, inward Ba2+ current through Ca2+ channels; IHC, inner hair cell; GFP, green fluorescent protein; IV, current-voltage relationship.

2 I. G. Huber, unpublished results.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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