IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2004;45:708-713.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0937
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (25)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Baumann, L.
Right arrow Articles by Wahl-Schott, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baumann, L.
Right arrow Articles by Wahl-Schott, C.

Functional Characterization of the L-type Ca2+ Channel Cav1.4{alpha}1 from Mouse Retina

Ludwig Baumann,1,2 Andrea Gerstner,1,2 Xiangang Zong,1 Martin Biel,1 and Christian Wahl-Schott1

1From the Department Pharmazie, Pharmakologie für Naturwissenschaften, Ludwig-Maximilians Universität Munich, Munich, Germany.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To study the electrophysiological and pharmacological properties of the L-type Ca2+ channel (LTCC) Cav1.4{alpha}1 ({alpha}1F) subunit from mouse retina and assess their contributions to the native retinal channel.

METHODS. The full-length cDNA of Cav1.4{alpha}1 was cloned from murine retina in an RT-PCR approach. Cav1.4{alpha}1 was expressed alone or together with the auxiliary {alpha}2{delta}1 and ß2a or ß3 subunits in HEK293 cells. The electrophysiological and pharmacological characteristics of L-type Ca2+ and Ba2+ inward currents (ICa and IBa) induced by Cav1.4{alpha}1 were determined by the whole-cell configuration of the patch-clamp method and compared with currents induced by the cardiac and smooth muscle-type Cav1.2{alpha}1 ({alpha}1C) channel.

RESULTS. Cav1.4{alpha}1-mediated IBa was observed only when the {alpha}2{delta}1 and ß subunits were coexpressed. Current densities were approximately two times higher with ß2a than with ß3. IBa activated faster and revealed much slower time-dependent inactivation than IBa induced by Cav1.2{alpha}1. Unlike in Cav1.2{alpha}1, inactivation was not accelerated with Ca2+ as the charge carrier, indicating the absence of Ca2+-dependent inactivation in Cav1.4{alpha}1. Cav1.4{alpha}1 exhibited voltage-dependent inactivation. The dihydropyridine (DHP) antagonist isradipine blocked Cav1.4{alpha}1 with approximately 20-fold lower sensitivity than Cav1.2{alpha}1. The agonistic DHP BayK 8644 stimulated maximum IBa approximately sixfold. Cav1.4{alpha}1 revealed only moderate sensitivities to L- and D-cis-diltiazem, with IC50 in the micromolar range. Both enantiomers unexpectedly blocked Cav1.4{alpha}1 with almost equal IC50.

CONCLUSIONS. The data indicate that Cav1.4{alpha}1 subunit constitutes the major molecular correlate of retinal L-type Ca2+ current. Its intrinsic biophysical properties, in particular its unique inactivation properties, enable Cav1.4{alpha}1 to provide a sustained ICa over a voltage range such as required for tonic glutamate release at the photoreceptor synapse.

The release of neurotransmitters from nerve terminals is initiated by Ca2+-influx through presynaptic voltage-dependent Ca2+ channels. Recent studies indicate that L-type Ca2+ channel (LTCCs) play a key role in the control of tonic glutamate release from retinal photoreceptors and bipolar cells.1 2 3 LTCCs recorded from photoreceptors4 5 6 7 and bipolar cells8 9 10 possess biophysical and pharmacological properties that set these channels apart from other LTCCs, such as cardiac and smooth muscle LTCCs. These unique properties include fast activation and very slow inactivation kinetics, as well as relatively low sensitivity to DHPs. LTCCs are not restricted to the synaptic layers of the retina. They have also been described in the cell soma of photoreceptors and in somatodendritic localization in bipolar and ganglion cells.10 11 12 These LTCCs are thought to supply the Ca2+ entry essential in more general cellular functions, such as Ca2+-mediated signal transduction or the regulation of gene transcription.13

LTCCs are multisubunit proteins consisting of the principal {alpha}1 and the auxiliary ß and {alpha}2{delta}14 15 subunits. Some LTCCs, such as the skeletal muscle Ca2+ channel contain an additional {gamma} subunit.14 Whereas {alpha}1 subunits determine the principal biophysical and pharmacological properties of the channel, ß subunits modulate cell surface expression, voltage dependence, and opening kinetics. So far, the functional role of the {alpha}2{delta} and the {gamma} subunit have been less well investigated.

Three different LTCC {alpha}1 subunits have been detected in the neuroretina, Cav1.2{alpha}1 ({alpha}1C), Cav1.3{alpha}1 ({alpha}1D), and Cav1.4{alpha}1 ({alpha}1F).12 In contrast to Cav1.2{alpha}1 and Cav1.3{alpha}1 which are expressed in a variety of tissues, Cav1.4{alpha}1 seems to be specifically expressed in the retina.16 17 Recently, mutations in the gene encoding Cav1.4{alpha}1 (CACNA1F) have been identified in patients who have incomplete X-linked congenital stationary night blindness16 17 18 19 (CSNB2). The key symptoms of this disease are impaired night vision and decreased visual acuity. The electrophysiological hallmark is the Schubert and Bornschein type electroretinogram, in which the amplitude of the scotopic b-wave is smaller than that of the normal sized a-wave. This finding suggests that the pathologic correlate of the disease is localized most likely at the photoreceptor-to-bipolar synapse.20 Although the genetic studies indicate that the functional loss of Cav1.4{alpha}1 causes CSNB2, the molecular mechanism by which mutations in this channel lead to disease is not understood. To address this important question in a physiological context, mouse models of this channelopathy are needed. To this end, we set out in this study to clone and functionally express the complete cDNA of Cav1.4{alpha}1 from mouse retina. The biophysical and pharmacological properties of Cav1.4{alpha}1-mediated calcium currents concurred well with those of calcium currents from photoreceptors and bipolar cells.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cloning of Murine Cav1.4{alpha}1
Total RNA was purified from retinas of C57Bl6 mice using the phenol-guanidine-isothiocyanate-chloroform extraction protocol (PeqGOLD Trifast; PeqLab, Erlangen, Germany). All animals were used in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. First-strand cDNA synthesis was performed with 5 µg total RNA, using oligo d(T) primer and reverse transcriptase (Superscript II; Invitrogen, Karlsruhe, Germany). To obtain the complete coding region of Cav1.4{alpha}1, three overlapping PCR fragments were amplified from the cDNA using the expand long template PCR system (Roche Diagnostics, Mannheim, Germany). The primers were designed according to the murine Cav1.4{alpha}1 sequence AF192497 (Table 1) . The PCR fragments were subcloned into pUC18 or pcDNA3 (Invitrogen, Karlsruhe, Germany) and several clones for each fragment were sequenced on both strands using the a chain terminator cycle sequencing kit (BigDye Terminator; Applied Biosystems, Foster City, CA). A eukaryotic expression vector for Cav1.4{alpha}1 was constructed by ligating the 3945-bp EcoRI/BamHI fragment (nucleotides [nt] 48-3992) containing an optimized sequence for initiation of translation and the 2013-bp BamHI/XhoI fragment (nt 3993-6005) into an EcoRI/SalI-cut bicistronic pIRES2-EGFP vector (Clontech, Heidelberg, Germany).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Sequences and Localization of PCR Primers Used for Cloning of Cav1.4{alpha}1

 
Heterologous Expression in HEK293 Cells and Electrophysiological Analysis of Cav1.4{alpha}1
HEK293 cells were transiently transfected with expression vectors encoding Cav1.4{alpha}1 or the Cav1.2{alpha}1, respectively, together with equimolar amounts of vectors encoding ß2a21 or ß321 and {alpha}2{delta}1,22 using transfection reagent (Fugene 6; Roche Diagnostics). For control experiments HEK293 cells were transfected with the empty enhanced green fluorescent protein (EGFP) vector. Cells were cultured in DMEM supplemented with 10% fetal calf serum and kept at 37°C, 10% CO2. ICa and IBa was measured from EGFP-positive cells using the following solutions (mM): (pipette solution) 112 CsCl, 3 MgCl2, 3 MgATP, 10 EGTA, and 5 HEPES, adjusted to pH 7.4 with CsOH; (bath solution) 82 NaCl, 30 BaCl2, 5.4 CsCl2, 1 MgCl2, 20 tetraethylammonium (TEA), and 5 HEPES, and 10 glucose, adjusted to pH 7.4 with NaOH. For experiments with 10 mM Ba2+ or 10 mM Ca2+ in the bath solution, the NaCl concentration was increased to 102 mM.

Currents were recorded at room temperature 2 to 4 days after transfection, using the whole-cell patch-clamp technique. Data were acquired at 10 kHz with an amplifier (Axopatch 200B with pClamp 8; Axon Instruments, Foster City, CA). Voltage-clamp data were stored on the computer hard disc and analyzed off-line (Clampfit 8; Axon Instruments, and Origin 6.1; OriginLab Co., Northampton, MA).

Patch pipettes were pulled from borosilicate glass and when filled with pipette solution, their input resistance was between 1.2 and 2.0 M{Omega}. Typical cell sizes were between 15 and 60 pF. Access resistances were between 3.0 and 4.0 M{Omega} and were compensated up to 70%. The holding potential was -80 mV, unless stated otherwise.

The peak I–V relationship was measured by applying 150-ms voltage pulses to potentials between -80 and +70 mV in 10-mV increments from a holding potential of -80 mV at 0.2 Hz. To obtain current densities, the current amplitude at Vmax was normalized to cell membrane capacitance (Cm). From I– V curves the activation threshold was determined as the test potential at which 5% of the maximum current was activated.

For determination of half-maximum activation voltage (V0.5,act) the chord conductance (G) was calculated from the current voltage curves by dividing the peak current amplitude by its driving force at that respective potential G = I/(V - Vrev), where Vrev is the extrapolated reversal potential, V is the membrane potential, and I is the peak current. The chord conductance was then fitted with a Boltzmann equation G = Gmax/(1 + e(V0.5,act-Vm)/kact), where Gmax is the maximum conductance, V0.5,act is the half-maximum activation voltage, Vm is the test potential, and kact is the slope factor of the activation curve.

For determination of half-maximum inactivation voltage (V0.5,inact) steady state inactivation curves were measured from a holding potential of -80 mV, by using a series of conditioning prepulses of different length to various voltages between -100 mV and +50 mV. For Cav1.2{alpha}1, the duration of the conditioning prepulse was 5 seconds, for Cav1.4{alpha}1 pulses of 5, 10, 20, and 30 seconds were applied to achieve steady state. The conditioning pulse was followed by a 20-ms return to the holding potential and a 300-ms test pulse to Vmax at 0.1 Hz or 0.05 Hz. Tail currents immediately after the final step to Vmax were normalized to maximum current amplitude and plotted as a function of the preceding membrane potential. The data points were fitted with the Boltzmann function: I = 1/(1 + e(Vm-V0.5,inact)/kinact) where Vm is the test potential, V0.5,inact is the half-maximum voltage for steady state inactivation, and k is the slope factor of the curve.

The time course of Cav1.2{alpha}1 current activation was fitted by the monoexponential function: It = A0 · e(-t/{tau}) + C, where It is the current at time t after a voltage pulse to Vmax, A0 is the steady state current amplitude with the respective time constant of activation, {tau}, and C is the remaining steady state current. Cav1.4{alpha}1 current activation was fitted by the biexponential function: It = Afast · e(-t/{tau}fast) + Aslow · e(-t/{tau}slow) + C, where {tau}slow and {tau}fast represent slow and fast time constants of activation, respectively.

To characterize the pharmacological properties of Cav1.4{alpha}1, we tested LTCC antagonists and the agonistic DHP BayK 8644. Drugs were applied by a local solution exchanger and reached the cell membrane within less than 100 ms. The effects of the antagonists were tested with 40-ms voltage-clamp steps to 0 mV or +10 mV from HPs of -80 mV or -50 mV. Pulse frequency was 0.2 Hz. For each test configuration, drug effects were measured after steady state block was attained within 2 to 3 minutes after drug application. For each antagonist the ratio Idrug/Icontrol was calculated from the peak current-voltage relations. The concentration-inhibition curve derived from responses at four to five different concentrations was obtained by fitting Idrug/Icontrol to the Hill equation 1/[1 + (IC50/C)]nH, where C is the drug concentration, nH is the Hill coefficient, and IC50 is the drug concentration needed for half-maximum block.

Stock solutions of drugs were prepared in H2O or ethanol (isradipine) and stored at +4°C in the dark. For electrophysiological measurements, stock solutions were freshly diluted in bath solution. Racemic verapamil HCl, D-cis-diltiazem, and S-(-)Bay K8644 were obtained from Sigma-Aldrich Corp. (St. Louis, MO), L-cis-diltiazem was purchased from Biomol Research Laboratories Inc. (Plymouth Meeting, PA), and racemic isradipine was a gift from Novartis Pharma AG (Basel, Switzerland).

Statistics
All results are expressed as the mean ± SEM; n is the number of experiments. An unpaired t-test was performed for the comparison between two groups. Significance was also tested by ANOVA, if multiple comparisons were made. P < 0.5 was considered significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Molecular Cloning of Murine Cav1.4{alpha}1
To clone murine Cav1.4{alpha}1 we designed specific primer pairs based on the previously published sequence of this channel (Table 1) 23 and performed RT-PCR with retinal cDNA from mouse strain C57Bl6. The full-length cDNA of Cav1.4{alpha}1 was determined to be 6111 bp with an open reading frame encoding a protein of 1984 amino acid residues (GenBank accession number of murine Cav1.4{alpha}1: AJ579852; available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). The sequence is nearly identical with the mouse sequence published by Naylor et al.23 with the notable exception of a strongly diverging sequence stretch in the C terminus (amino acid residues 1768-1807). Inspection of the nucleotide sequences revealed that the profound differences in this stretch arise from frame shifts in the cDNA. The strong homology between the sequence published in this study to that of rat and human Cav1.4{alpha}116 17 20 (see sequence alignment in Supplemental Figure 1, available online at http://www.iovs.org/cgi/content/full/45/2/708/DC1) strongly indicates that our sequence version represents the correct one.

Electrophysiological Properties of Murine Cav1.4{alpha}1
HEK293 cells transfected with Cav1.4{alpha}1 revealed a voltage-dependent IBa that was not different from IBa of control cells transfected with an empty vector (current densities at Vmax in 30 mM Ba2+ Cav1.4{alpha}1: -1.2 ± 0.2 pA/pF, n = 12; empty vector: -1.3 ± 0.3 pA/pF, n = 7). This finding indicated that Cav1.4{alpha}1 was not able to induce the formation of a functional LTCC. In contrast, when Cav1.4{alpha}1 was coexpressed with {alpha}2{delta}1 and ß2a subunits, amplitudes of IBa consistently exceeded those of the endogenous current. In addition, the biophysical properties of the heterologously expressed current were clearly different from those of the endogenous current. Figure 1A shows representative Ba2+ current traces of Cav1.4{alpha}1 in comparison to current traces of the smooth muscle and cardiac type Cav1.2{alpha}1. It is evident that Cav1.4{alpha}1 activated faster ({tau}fast: 0.65 ± 0.04 ms; {tau}slow: 3.6± 0.58 ms; n = 24; relative contribution of slow component: 0.35 ± 0.05) than Cav1.2{alpha}1 ({tau}: 1.59 ± 0.47 ms, n = 8). Moreover, Cav1.4{alpha}1 displayed extremely slow inactivation kinetics. During the 150-ms voltage step shown in Figure 1A the current did not significantly decrease. At +10 mV it took more than 30 seconds for full inactivation (not shown). Consistent with the properties of an LTCC Cav1.4{alpha}1 activated at relatively positive membrane potentials. In experiments performed with 30 mM Ba2+ as the charge carrier, the mean I– V relationships of Cav1.4{alpha}1 and Cav1.2{alpha}1 were almost identical (Fig. 1B) . The threshold for Cav1.4{alpha}1 current activation was -28 ± 1.2 mV (n = 30), which is 5 mV more negative than Cav1.2{alpha}1 current activation (-23.4 ± 1.9 mV; n = 9). For both {alpha}1 subunits, the peak current occurred at similar Vmax with 13.8 ± 0.9 mV (n = 30) for Cav1.4{alpha}1 and 13.3 ± 1.7 mV (n = 9) for Cav1.2{alpha}1 (Fig. 1B) . The peak current densities for Cav1.4{alpha}1 was -9.5 ± 1.1 pA/pF (n = 30) and -31.4 ± 9.4 pA/pF (n = 9) for Cav1.2{alpha}1. To compare the voltage-dependent activation and inactivation of the Cav1.4{alpha}1 and Cav1.2{alpha}1 currents, normalized conductance-voltage relations and steady state inactivation curves of IBa were determined and fitted by Bolzmann distributions (Fig. 1C) . The activation curves for both {alpha}1 subunits were almost identical. The potential of half-maximum IBa activation (V0.5,act) was 1.1 ± 1.0 mV for Cav1.4{alpha}1 (n = 28) and -0.1 ± 1.2 mV for Cav1.2{alpha}1 (n = 9). In contrast, at a conditioning pulse duration of 5 seconds, the steady state inactivation curve of Cav1.4{alpha}1 was shifted to approximately 20 mV more depolarized potentials with respect to Cav1.2{alpha}1. The potential of half-maximum IBa inactivation (V0.5,inact) was 0.64 ± 2.6 mV for the Cav1.4{alpha}1 subunit (n = 9) and -24.3 ± 1.5 mV for the Cav1.2{alpha}1 subunit (n = 9).



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 1. Cav1.4{alpha}1 induces L-type IBa in HEK293 cells. For all experiments Cav1.4{alpha}1 or Cav1.2{alpha}1 was coexpressed with {alpha}2{delta}1 and ß2a. Currents were measured in bath solution containing 30 mM Ba2+ as the charge carrier. Biophysical parameters are given in Table 2 . (A) Whole-cell current recorded from representative cells expressing either Cav1.4{alpha}1 (left) or Cav1.2{alpha}1 (right). Currents were recorded from an HP of -80 mV by applying 150-ms pulses to membrane voltages between -80 mV and +70 mV at 0.2 Hz. (B) I– V relationship for Cav1.4{alpha}1 channels (•; n = 30) and Cav1.2{alpha}1 ({circ}; n = 9). Individual I– V curves were normalized to the respective maximum current amplitude and then averaged. (C) Conductance-voltage relationships for Cav1.4{alpha}1 (•; n = 28) and Cav1.2{alpha}1 ({circ}; n = 9) and steady state inactivation curves for Cav1.4{alpha}1 ({blacksquare}; n = 9) and Cav1.2{alpha}1 ({square}; n = 9) were determined. Individual curves were normalized to maximum current amplitude and then averaged. Solid lines: fits of the data to the Boltzmann equation. Inactivation curves were determined using a conditioning prepulse of 5 seconds and a 300-ms test pulse to +10 mV.

 
Voltage-dependent inactivation of Cav1.4{alpha}1 was dependent on the length of the conditioning prepulse. Increasing the pulse duration from 5 to 30 seconds increased kinact of the inactivation curve and shifted V0.5,inact to approximately 25 mV more hyperpolarized voltages (Fig. 2) . There was no significant difference between the inactivation curves for the 20-second and 30-second prepulse duration, indicating that steady state was achieved after 20 seconds.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 2. Dependence of steady inactivation of Cav1.4{alpha}1 on prepulse duration. Ba2+ (30 mM) was used as the charge carrier. The following parameters were obtained: 5-second prepulse: V0.5,inact = 0.64 ± 2.6 mV, kinact = 17.5 ± 0.8, n = 9; 10-second prepulse: V0.5,inact = -20.1 ± 1.6 mV, kinact = 14.5 ± 1.0 mV, n = 8; 20-second prepulse: V0.5,inact = -21.4 ± 2.1mV, kinact = 10.1 mV ± 1.0, n = 7; 30-second prepulse: V0.5,inact = -25.7 ± 1.9 mV, kinact = 8.12 mV ± 0.6, n = 6.

 
Cav1.4{alpha}1 also formed functional channels with the ß3 subunit, an auxiliary LTCC subunit that is expressed in the central nervous system (CNS) and in a variety of peripheral tissues.15 Peak current densities obtained after coexpression of Cav1.4{alpha}1 + ß3 + {alpha}2{delta}1 were less than half those recorded in the presence of ß2a (ß3: -4.4 ± 0.8 pA/pF, n = 10; ß2a: -9.5 ± 1.1 pA/pF, n = 30). However, the biophysical properties of both currents were very similar to each (Table 2) . As the only exception, the activation threshold was slightly shifted to more hyperpolarized voltages in the presence of ß3 (ß2a: -28.0 ± 1.2 mV, n = 30; ß3: -37.2 ± 2.4 mV, n = 10). Ca2+ is not only the permeating cation of LTCCs, it is also a key determinant of their inactivation. Figure 3A shows current traces of Cav1.4{alpha}1/ß2a/{alpha}2{delta}1 in the presence of 10 mM extracellular Ba2+ or Ca2+, respectively. Both current traces show identical kinetics, indicating that unlike in the Cav1.2{alpha}1 channel,24 Ca2+ does not accelerate inactivation. However, the current density of ICa was reduced by 30.1% ± 0.03% (n = 9) in comparison to IBa. In addition, 10 mM Ca2+ as the charge carrier shifted the I– V relation to approximately 10 mV more positive voltages without affecting the slope of the activation and inactivation curves (Fig. 3B , Table 2 ).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Biophysical Properties of IBa and ICa through Heterologously Expressed Cav1.4{alpha}1 Subunits

 


View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 3. (A) Current traces recorded from a Cav1.4{alpha}1/ß2/{alpha}2{delta}1-expressing cell with either 10 mCa2+ (ICa) or 10 mM Ba2+ (IBa) as the charge carrier. Currents were evoked from an HP of -80 mV by applying 200-ms voltage steps to +10 mV at 0.2 Hz. There was no current inactivation in both cases. (B) Normalized I– V relationship for Cav1.4{alpha}1 recorded in bath solution containing 10 mM Ca2+ ({circ}; n = 9) or Ba2+ (•; n = 16).

 
Pharmacological Characterization of Cav1.4{alpha}1
To characterize the pharmacological profile of Cav1.4{alpha}1 we quantified the blocking effect of the Ca2+ channel antagonists isradipine, verapamil, L-cis-diltiazem, and D-cis-diltiazem on IBa. At -80 mV the DHP antagonist isradipine blocked IBa elicited by depolarizing pulses to Vmax given at 0.2 Hz with an apparent IC50 of 200 ± 50 nM (n = 5–9; Fig. 4A ). At a holding potential of -80 mV 100 nM isradipine blocked 41.9% ± 0.03% (n = 7) of IBa. Changing the holding potential to -50 mV significantly increased the block to 88.3% ± 0.01% (n = 9) of IBa, indicating a strong voltage-dependence of the observed block (Fig. 4A) . Cav1.4{alpha}1 was only weakly sensitive to verapamil. At an HP of -80 mV 100 µM verapamil blocked 69.1% ± 0.13% (n = 5) of IBa.



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 4. Sensitivity of Cav1.4{alpha}1 for LTCC blockers and BayK 8644. For all experiments Cav1.4{alpha}1 was coexpressed with {alpha}2{delta}1 and ß2a. Currents were measured in bath solution containing 30 mM Ba2+ as the charge carrier. (A) Concentration-response curves for inhibition of Cav1.4{alpha}1 by D-cis-diltiazem ({triangledown}; n = 5–6), L-cis-diltiazem ({blacktriangledown}; n = 5–6), and isradipine (•; n = 5–9). Pronounced voltage dependence of isradipine block was observed when HP was changed from -80 mV to -50 mV ({circ}; n = 9). (B) I– V relationship for Cav1.4{alpha}1 in the absence (•; n = 30) and presence of 1 µM Bay K 8644 ({circ}; n = 5).

 
We also tested the blocking effect of the two enantiomers of diltiazem under identical conditions. Unexpectedly, the concentration–response relationships were not statistically different from each other (Fig. 4A) . L-cis-Diltiazem blocked IBa with an IC50 of 74.8 ± 8.3 µM (n = 4–6), and D-cis-diltiazem blocked IBa with an IC50 of 91.6 ± 9.4 µM (n = 4–6). This observation is very surprising, because the affinity of L-cis-diltiazem for LTCCs is usually several orders of magnitude lower than that of D-cis-diltiazem.25

Finally, we tested the effect of the DHP agonist BayK 8644 on Cav1.4{alpha}1. At a concentration of 1 µM this substance increased the current density of IBa approximately sixfold. As in other LTCCs, BayK 8644 shifted the I– V relationship to approximately 8 to 10 mV more hyperpolarized potentials (Fig. 4B , Table 2 ).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study we report the cloning and the functional characterization of the Cav1.4{alpha}1 calcium channel from mouse retina. Cav1.4{alpha}1 requires the coexpression of auxiliary ß and {alpha}2{delta} subunits to yield calcium currents in HEK293 cells. This finding indicates that Cav1.4{alpha}1, like other members of the LTCC family forms a functional multisubunit complex in the plasma membrane. Currents obtained on coexpression with ß2 had consistently bigger amplitudes than currents obtained with ß3. One explanation for this finding is that although Cav1.4{alpha}1 principally can assemble with different ß subunits, it preferentially binds to ß2. The key role of the ß2 subunit is also demonstrated by a recent study showing that elimination of this subunit in mouse retina produces a phenotype similar to CSNB2 in humans.26 In contrast, no retinal phenotype has been reported so far for ß3-27 and ß4-deficient mice.28 Taken together, these findings strongly indicate that the ß2 subunit is obligatorily required for the formation of native retinal LTCCs.

Cav1.4{alpha}1 possesses unique biophysical and pharmacological properties that set this channel apart from the cardiac and smooth muscle Cav1.2{alpha}1. Currents induced by Cav1.4{alpha}1 activate with very fast kinetics, but display an extremely slow time course of inactivation. At Vmax, inactivation of IBa requires many seconds. Inactivation of Cav1.4{alpha}1 is not accelerated in the presence of extracellular Ca2+. Typical LTCCs such as Cav1.2{alpha}1 are strongly regulated by the passage of Ca2+ through the channel pore. Entering Ca2+ interacts with calmodulin bound to the carboxyl terminus of the channel, thereby causing a decay of the current within milliseconds.14 24 The lack of Ca2+-dependent inactivation in Cav1.4{alpha}1 indicates that either the binding of calmodulin itself or the conformational steps coupling Ca2+-binding to channel inactivation are impaired in this channel. The C-terminal sequences conferring Ca2+-dependent inactivation in Cav1.2{alpha}24 29 30 31 32 33 34 35 are highly conserved in Cav1.4{alpha}. It is not known whether the few amino acid exchanges in these sequences explain the different inactivation properties of both channels. Alternatively, not yet identified channel domains may cause the lack of Ca2+-dependent inactivation in Cav1.4{alpha}1. In the absence of the Ca2+-dependent mechanism the time course of currents through Cav1.4{alpha}1 is determined primarily by voltage-dependent inactivation, causing the extremely slow current decay typical of Cav1.4{alpha}1.

Cav1.4{alpha}1 channels reveal a unique pharmacological profile. As for native retinal LTCCs, the apparent DHP antagonist sensitivity of Cav1.4{alpha}1 is approximately 20-fold lower than for the cardiac and smooth muscle Cav1.2{alpha}1 at -80 mV. Twelve of 13 amino acid residues required for high DHP sensitivity in Cav1.2{alpha}1 are present in the primary sequence of Cav1.4{alpha}1.36 As the only difference, Cav1.4{alpha}1 contains a phenylalanine at position 1414 instead of the tyrosine found in Cav1.2{alpha}1. However, this exchange cannot account for the low affinity because Cav1.3{alpha}1, a channel with a DHP sensitivity similar to that of Cav1.4{alpha}1,37 contains at the equivalent position a tyrosine, as does Cav1.2{alpha}1. Although it cannot be excluded that there are other amino acids not yet identified that determine apparent affinities, it is probable that the differences reflect the profound voltage-dependence of the DHP block. In contrast to Cav1.2{alpha}1, Cav1.4{alpha}1 inactivates very slowly at negative holding potentials. Because DHPs bind preferentially to the inactivated state of LTCCs a decrease of apparent affinity is caused by this behavior. Indeed, shifting the holding potential from -80 to -50 mV, hence increasing the fraction of channels being in the inactivated state, strongly increased the DHP block of IBa. The specific inactivation properties could also underlie the relatively low sensitivities of Cav1.4{alpha}1 currents to verapamil and diltiazem. Unexpectedly, L-cis-diltiazem and D-cis-diltiazem blocked IBa through Cav1.4{alpha}1 with almost the same apparent IC50. In other LTCCs the affinity of D-cis-diltiazem is at least 20 times higher than that of L-cis-diltiazem.25 38 39 In Cav1.2{alpha}1 the binding site for diltiazem is overlapping with that for DHPs.40 With the exception of the Y1414F exchange the identified amino acids are completely represented in Cav1.4{alpha}1, indicating that other channel domains and/or a differential voltage dependence of the block by the D and L enantiomer of diltiazem determine the observed difference. Micromolar concentrations of L-cis-diltiazem are commonly used to block Ca2+ flux through cyclic nucleotide-gated (CNG) channels in rod and cone photoreceptors.41 42 Because comparable concentrations also block calcium currents through Cav1.4{alpha}1, extreme care is necessary to distinguish the effects of this blocker on CNG channels from those on retinal LTCCs.

In summary, currents through heterologously expressed Cav1.4{alpha}1/ß2a/a2{delta}1 closely resemble the slowly inactivating Ca2+ and Ba2+ currents observed in native photoreceptors4 5 6 and bipolar cells.8 9 As the only difference, Ca2+ currents from native photoreceptors and bipolar cells activate at 10 to 15 mV more negative potentials4 5 6 9 10 than currents obtained after heterologous expression of Cav1.4{alpha}1. Several factors, such as the absence of auxiliary channel subunits, cytosolic modulators or posttranslational modifications in HEK293 cells, may explain the discrepancy between native and expressed channels. Nevertheless, its unique electrophysiological properties predestine Cav1.4{alpha}1 to fulfill the specific tasks required for normal retinal function. In particular, the slow inactivation of Cav1.4{alpha}1 is well suited to mediate tonic glutamate release from synaptic terminals of ribbon synapses in photoreceptors and bipolar cells.9 The properties of mouse Cav1.4{alpha}1 are consistent with those of the heterologously expressed human Cav1.4{alpha}1.43 For example, both channels lack Ca2+-dependent inactivation, assemble with ß-subunits and reveal intermediate DHP sensitivity. The human Cav1.4{alpha}1 activates at a slightly more negative voltage than the murine Cav1.4{alpha}1. The small difference may be attributed to an intrinsic species difference between mouse and humans or, alternatively, may be due to the slightly different experimental setup used to study both currents. Taken together, our results indicate that mice deficient in this channel will be a useful in vivo model for studying CSNB2.


   Acknowledgements
 
The authors thank Norbert Klugbauer for the gift of Cav1.2{alpha}1, {alpha}2{delta}1, ß2a, and ß3 expression vectors.


   Footnotes
 
2 Contributed equally to the work and therefore should be considered equivalent senior authors. Back

Supported by grants from Deutsche Forschungsgemeinschaft.

Submitted for publication August 27, 2003; revised October 16, 2003; accepted October 22, 2003.

Disclosure: L. Baumann, None; A. Gerstner, None; X. Zong, None; M. Biel, None; C. Wahl-Schott, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Christian Wahl-Schott, Department Pharmazie, Pharmakologie für Naturwissenschaften, Ludwig-Maximilians Universität München, Butenandtstr. 7, 81377 München, Germany; christian.wahl{at}cup.uni-muenchen.de.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tachibana M, Okada T, Arimura T, Kobayashi K, Piccolino M. Dihydropyridine-sensitive calcium current mediates neurotransmitter release from bipolar cells of the goldfish retina. J Neurosci. 1993;13:2898–2909.[Abstract]
  2. Schmitz Y, Witkovsky P. Dependence of photoreceptor glutamate release on a dihydropyridine-sensitive calcium channel. Neuroscience. 1997;78:1209–1216.[CrossRef][ISI][Medline][Order article via Infotrieve]
  3. Rieke F, Schwartz EA. Asynchronous transmitter release: control of exocytosis and endocytosis at the salamander rod synapse. J Physiol. 1996;493:1–8.[ISI][Medline][Order article via Infotrieve]
  4. Wilkinson MF, Barnes S. The dihydropyridine-sensitive calcium channel subtype in cone photoreceptors. J Gen Physiol. 1996;107:621–630.[Abstract/Free Full Text]
  5. Taylor WR, Morgans C. Localization and properties of voltage-gated calcium channels in cone photoreceptors of Tupaia belangeri. Vis Neurosci. 1998;15:541–552.[CrossRef][ISI][Medline][Order article via Infotrieve]
  6. Kourennyi DE, Barnes S. Depolarization-induced calcium channel facilitation in rod photoreceptors is independent of G proteins and phosphorylation. J Neurophysiol. 2000;84:133–138.[Abstract/Free Full Text]
  7. Stella SL, Jr, Bryson EJ, Thoreson WB. A2 adenosine receptors inhibit calcium influx through L-type calcium channels in rod photoreceptors of the salamander retina. J Neurophysiol. 2002;87:351–360.[Abstract/Free Full Text]
  8. von Gersdorff H, Matthews G. Calcium-dependent inactivation of calcium current in synaptic terminals of retinal bipolar neurons. J Neurosci. 1996;16:115–122.[Abstract/Free Full Text]
  9. Protti DA, Llano I. Calcium currents and calcium signaling in rod bipolar cells of rat retinal slices. J Neurosci. 1998;18:3715–3724.[Abstract/Free Full Text]
  10. Berntson A, Taylor WR, Morgans CW. Molecular identity, synaptic localization, and physiology of calcium channels in retinal bipolar cells. J Neurosci Res. 2003;71:146–151.[CrossRef][ISI][Medline][Order article via Infotrieve]
  11. Morgans CW. Localization of the {alpha}1F calcium channel subunit in the rat retina. Invest Ophthalmol Vis Sci. 2001;42:2414–2418.[Abstract/Free Full Text]
  12. Xu HP, Zhao JW, Yang XL. Expression of voltage-dependent calcium channel subunits in the rat retina. Neurosci Lett. 2002;329:297–300.[CrossRef][ISI][Medline][Order article via Infotrieve]
  13. Firth SI, Morgan IG, Boelen MK, Morgans CW. Localization of voltage-sensitive L-type calcium channels in the chicken retina. Clin Exp Ophthalmol. 2001;29:183–187.[CrossRef][ISI][Medline][Order article via Infotrieve]
  14. Catterall WA. Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell Dev Biol. 2000;16:521–555.[CrossRef][ISI][Medline][Order article via Infotrieve]
  15. Hofmann F, Lacinova L, Klugbauer N. Voltage-dependent calcium channels: from structure to function. Rev Physiol Biochem Pharmacol. 1999;139:33–87.[ISI][Medline][Order article via Infotrieve]
  16. Strom TM, Nyakatura G, Apfelstedt-Sylla E, et al. An L-type calcium-channel gene mutated in incomplete X-linked congenital stationary night blindness. Nat Genet. 1998;19:260–263.[CrossRef][ISI][Medline][Order article via Infotrieve]
  17. Bech-Hansen NT, Naylor MJ, Maybaum TA, et al. Loss-of-function mutations in a calcium-channel alpha1-subunit gene in Xp11.23 cause incomplete X-linked congenital stationary night blindness. Nat Genet. 1998;19:264–267.[CrossRef][ISI][Medline][Order article via Infotrieve]
  18. Wutz K, Sauer C, Zrenner E, et al. Thirty distinct CACNA1F mutations in 33 families with incomplete type of XLCSNB and Cacna1f expression profiling in mouse retina. Eur J Hum Genet. 2002;10:449–456.[CrossRef][ISI][Medline][Order article via Infotrieve]
  19. Boycott KM, Maybaum TA, Naylor MJ, et al. A summary of 20 CACNA1F mutations identified in 36 families with incomplete X-linked congenital stationary night blindness, and characterization of splice variants. Hum Genet. 2001;108:91–97.[CrossRef][ISI][Medline][Order article via Infotrieve]
  20. Morgans CW, Gaughwin P, Maleszka R. Expression of the alpha1F calcium channel subunit by photoreceptors in the rat retina. Mol Vis. 2001;7:202–209.[ISI][Medline][Order article via Infotrieve]
  21. Hullin R, Singer-Lahat D, Freichel M, et al. Calcium channel beta subunit heterogeneity: functional expression of cloned cDNA from heart, aorta and brain. EMBO J. 1992;11:885–890.[ISI][Medline][Order article via Infotrieve]
  22. Ellis SB, Williams ME, Ways NR, et al. Sequence and expression of mRNAs encoding the alpha 1 and alpha 2 subunits of a DHP-sensitive calcium channel. Science. 1988;241:1661–1664.[Abstract/Free Full Text]
  23. Naylor MJ, Rancourt DE, Bech-Hansen NT. Isolation and characterization of a calcium channel gene, Cacna1f, the murine orthologue of the gene for incomplete X-linked congenital stationary night blindness. Genomics. 2000;66:324–327.[CrossRef][ISI][Medline][Order article via Infotrieve]
  24. Budde T, Meuth S, Pape HC. Calcium-dependent inactivation of neuronal calcium channels. Nat Rev Neurosci. 2002;3:873–883.[CrossRef][ISI][Medline][Order article via Infotrieve]
  25. Glossmann H, Linn T, Rombusch M, Ferry DR. Temperature-dependent regulation of d-cis-[3H]diltiazem binding to Ca2+ channels by 1,4-dihydropyridine channel agonists and antagonists. FEBS Lett. 1983;160:226–232.[CrossRef][ISI][Medline][Order article via Infotrieve]
  26. Ball SL, Powers PA, Shin HS, Morgans CW, Peachey NS, Gregg RG. Role of the ß2 subunit of voltage-dependent calcium channels in the retinal outer plexiform layer. Invest Ophthalmol Vis Sci. 2002;43:1595–1603.[Abstract/Free Full Text]
  27. Namkung Y, Smith SM, Lee SB, et al. Targeted disruption of the Ca2+ channel beta3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltage-dependent activation of P/Q-type Ca2+ channels in neurons. Proc Natl Acad Sci USA. 1998;95:12010–12015.[Abstract/Free Full Text]
  28. Burgess DL, Jones JM, Meisler MH, Noebels JL. Mutation of the Ca2+ channel beta subunit gene Cchb4 is associated with ataxia and seizures in the lethargic (lh) mouse. Cell. 1997;88:385–392.[CrossRef][ISI][Medline][Order article via Infotrieve]
  29. Qin N, Olcese R, Bransby M, Lin T, Birnbaumer L. Ca2+-induced inhibition of the cardiac Ca2+ channel depends on calmodulin. Proc Natl Acad Sci USA. 1999;96:2435–2438.[Abstract/Free Full Text]
  30. Pitt GS, Zuhlke RD, Hudmon A, Schulman H, Reuter H, Tsien RW. Molecular basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels. J Biol Chem. 2001;276:30794–30802.[Abstract/Free Full Text]
  31. Peterson BZ, Lee JS, Mulle JG, Wang Y, de Leon M, Yue DT. Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels. Biophys J. 2000;78:1906–1920.[Abstract/Free Full Text]
  32. Mouton J, Feltz A, Maulet Y. Interactions of calmodulin with two peptides derived from the c-terminal cytoplasmic domain of the Ca(v)1.2 Ca2+ channel provide evidence for a molecular switch involved in Ca2+-induced inactivation. J Biol Chem. 2001;276:22359–22367.[Abstract/Free Full Text]
  33. Zuhlke RD, Pitt GS, Deisseroth K, Tsien RW, Reuter H. Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature. 1999;399:159–162.[CrossRef][Medline][Order article via Infotrieve]
  34. Pate P, Mochca-Morales J, Wu Y, et al. Determinants for calmodulin binding on voltage-dependent Ca2+ channels. J Biol Chem. 2000;275:39786–39792.[Abstract/Free Full Text]
  35. Romanin C, Gamsjaeger R, Kahr H, et al. Ca(2+) sensors of L-type Ca(2+) channel. FEBS Lett. 2000;487:301–306.[CrossRef][ISI][Medline][Order article via Infotrieve]
  36. Striessnig J, Grabner M, Mitterdorfer J, Hering S, Sinnegger MJ, Glossmann H. Structural basis of drug binding to L Ca2+ channels. Trends Pharmacol Sci. 1998;19:108–115.[CrossRef][Medline][Order article via Infotrieve]
  37. Xu M, Welling A, Paparisto S, Hofmann F, Klugbauer N. Enhanced expression of L-type Cav1.3 calcium channels in murine embryonic hearts from Cav1.2 deficient mice. J Biol Chem. 2003;278:40837–40841.[Abstract/Free Full Text]
  38. Bohle T. The effect of the benzothiazepine diltiazem on force and Ca2+ current in isolated frog skeletal muscle fibres. J Physiol. 1992;445:303–318.[Abstract/Free Full Text]
  39. Hockerman GH, Peterson BZ, Johnson BD, Catterall WA. Molecular determinants of drug binding and action on L-type calcium channels. Annu Rev Pharmacol Toxicol. 1997;37:361–396.[CrossRef][ISI][Medline][Order article via Infotrieve]
  40. Hockerman GH, Dilmac N, Scheuer T, Catterall WA. Molecular determinants of diltiazem block in domains IIIS6 and IVS6 of L-type Ca(2+) channels. Mol Pharmacol. 2000;58:1264–1270.
  41. Kaupp UB, Seifert R. Cyclic nucleotide-gated ion channels. Physiol Rev. 2002;82:769–824.[Abstract/Free Full Text]
  42. Finn JT, Grunwald ME, Yau KW. Cyclic nucleotide-gated ion channels: an extended family with diverse functions. Annu Rev Physiol. 1996;58:395–426.[CrossRef][ISI][Medline][Order article via Infotrieve]
  43. Koschak A, Reimer D, Walter D, et al. Cav1.4alpha1 subunits can form slowly inactivating dihydropyridine-sensitive L-type Ca2+ channels lacking Ca2+-dependent inactivation. J Neurosci. 2003;23:6041–6049.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
C. Wahl-Schott, L. Baumann, H. Cuny, C. Eckert, K. Griessmeier, and M. Biel
Switching off calcium-dependent inactivation in L-type calcium channels by an autoinhibitory domain
PNAS, October 17, 2006; 103(42): 15657 - 15662.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
G. Baroudi, Y. Qu, O. Ramadan, M. Chahine, and M. Boutjdir
Protein kinase C activation inhibits Cav1.3 calcium channel at NH2-terminal serine 81 phosphorylation site.
Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1614 - H1622.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
C. J. Doering, J. Hamid, B. Simms, J. E. McRory, and G. W. Zamponi
Cav1.4 Encodes a Calcium Channel with Low Open Probability and Unitary Conductance
Biophys. J., November 1, 2005; 89(5): 3042 - 3048.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
F. Mansergh, N. C. Orton, J. P. Vessey, M. R. Lalonde, W. K. Stell, F. Tremblay, S. Barnes, D. E. Rancourt, and N. T. Bech-Hansen
Mutation of the calcium channel gene Cacna1f disrupts calcium signaling, synaptic transmission and cellular organization in mouse retina
Hum. Mol. Genet., October 15, 2005; 14(20): 3035 - 3046.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurophysiol.Home page
D. Cia, A. Bordais, C. Varela, V. Forster, J. A. Sahel, A. Rendon, and S. Picaud
Voltage-Gated Channels and Calcium Homeostasis in Mammalian Rod Photoreceptors
J Neurophysiol, March 1, 2005; 93(3): 1468 - 1475.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
J.-C. Hoda, F. Zaghetto, A. Koschak, and J. Striessnig
Congenital Stationary Night Blindness Type 2 Mutations S229P, G369D, L1068P, and W1440X Alter Channel Gating or Functional Expression of Cav1.4 L-type Ca2+ Channels
J. Neurosci., January 5, 2005; 25(1): 252 - 259.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
P. Koulen, J. Liu, E. Nixon, and C. Madry
Interaction between mGluR8 and Calcium Channels in Photoreceptors Is Sensitive to Pertussis Toxin and Occurs Via G Protein {beta}{gamma} Subunit Signaling
Invest. Ophthalmol. Vis. Sci., January 1, 2005; 46(1): 287 - 291.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurophysiol.Home page
D. Lipscombe, T. D. Helton, and W. Xu
L-Type Calcium Channels: The Low Down
J Neurophysiol, November 1, 2004; 92(5): 2633 - 2641.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow