©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Involvement of the Carboxyl-terminal Region of the Subunit in Voltage-dependent Inactivation of Cardiac Calcium Channels (*)

(Received for publication, May 5, 1995)

Udo Klöckner (1), Gabor Mikala (2)(§), Maria Varadi (2), Gyula Varadi (2)(¶), Arnold Schwartz (2)

From the  (1)Department of Physiology, University of Cologne, Robert-Kochstrasse 39, 50931 Cologne, Germany and the (2)Institute of Molecular Pharmacology and Biophysics, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0828

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Intracellular application of proteases increases cardiac calcium current to a level similar to -adrenergic stimulation. Using transiently transfected HEK 293 cells, we studied the molecular mechanism underlying calcium channel stimulation by proteolytic treatment. Perfusion of HEK cells, coexpressing the human cardiac (hHT) , and subunits, with 1 mg/ml of trypsin or carboxypeptidase A, increased the peak amplitude of the calcium channel current 3-4-fold without affecting the voltage dependence. Similar results were obtained in HEK cells cotransfected with hHT and or with alone, suggesting that modification of the subunit itself is responsible for the current enhancement by proteolysis. To further characterize the modification of the subunit by trypsin, we expressed a deletion mutant in which part of the carboxyl-terminal tail up to amino acid 1673 was removed. The expressed calcium channel currents no longer responded to intracellular application of the proteases; however, a 3-fold higher current density as well as faster inactivation compared with the wild type was observed. The results provide evidence that a specific region of the carboxyl-terminal tail of the cardiac subunit is an important regulatory segment that may serve as a critical component of the gating machinery that influences both inactivation properties as well as channel availability.


INTRODUCTION

Voltage-dependent L-type calcium channels play a major role in excitation-contraction coupling of cardiac muscle. One of the physiologically important features of these channels is their ability to respond to -adrenergic stimulation with enhanced activity, contributing to an increase in cardiac contractility. There is accumulating evidence that the increase in activity is due to phosphorylation of the calcium channel protein itself or a closely associated protein ( (1) and references therein). The loci of the channel proteins that may be involved in this stimulation are as yet unknown and in fact are controversial(2, 3, 4, 5, 6, 7) .

It was shown some years ago that the major structural determinants of voltage-gated ion channels are accessible to internally perfused proteases, leading to modification in function(8, 9, 10, 11, 12) . In cardiac myocytes, intracellular application of trypsin dramatically increased the amplitude of the calcium current (I)(13) . Addition of isoproterenol, subsequent to trypsin treatment, did not further increase the current amplitude, and the enhancement of I could not be blocked by inhibitors of cAMP-dependent phosphorylation(13) . In an obverse experiment, I up-modulated by isoproterenol was insensitive to trypsin. From these findings, the authors (13) speculated that trypsin removed an ``inactivation gate'' that is normally controlled by phosphorylation. However, up to now no structural proof of this hypothesis is available. Furthermore, the action of trypsin on the cardiac L-type calcium channels at the molecular level is unclear. In view of the accepted heterooligomeric structure of the cardiac calcium channel complex, i.e. there are four subunits(2, 14) , proteolytic modification could modify any and all of the subunits. In the present study, using transiently transfected HEK293 cells, we have demonstrated that intracellular trypsin or carboxypeptidase A treatment functionally modifies primarily a specific region of the carboxyl terminus of the subunit.


EXPERIMENTAL PROCEDURES

Construction of the Deletion Mutant hHT-1673

A cassette harboring the altered region of the hHT-1 clone (15) was constructed using the polymerase chain reaction (Roche Molecular Systems) mutagenesis procedure. Preparative amplifications utilized the following primers for 20 cycles: 5`-GGTGCCCCCTGCAGGTG-3` and 5`-GTCTAGACCTACAGGCCACCGGCCCTCCTG-3`. The restriction sites Sse8387I and XbaI are underlined, respectively. The amber stop codon introduced is shown in boldfacetype. The amplification product was isolated from a 2% sieving agarose gel, subcloned into pBluescript SK(+) via blunt end ligation, and was sequenced. The verified cassette was then used to replace the Sse8387I(6143)-XbaI(polylinker) fragment of hHT-1.

Isolation of the H cDNA Clone

Reverse transcription followed by polymerase chain reaction was used to isolate a clone encoding the subunit from human cardiac mRNA. Briefly, total RNA was isolated from surgically removed human cardiac samples by the acidic guanidium isothiocyanate method(16) . Poly(A) RNA was isolated by the standard oligo(dT)-cellulose chromatography(17) . Reverse transcription was done using 200 ng of poly(A) RNA, oligo(dT) as primer, and 100 units of SuperScript reverse transcriptase (Life Technologies, Inc.), according to the manufacturer's protocol. The resultant first strand cDNA pool was subjected to polymerase chain reaction according to the suggestions of the manufacturer of the Vent polymerase. After denaturing and heat inactivation of the reverse transcriptase at 95 °C for 3 min, the sample was subjected to 39 cycles of thermal cycling at 60 °C for 1 min followed by 72 °C for 1 min. The primers were: 5`-CCCATGTATGACGACTCCTACC-3` and 5`-GCAGGAGGCTGTCAGTAGCTATCC-3`. The 1.4-kilobase polymerase chain reaction product was isolated from a preparative agarose gel, subcloned into pBluescript SK(+) via blunt end ligation to the EcoRV site, and was sequenced. The sequence was found to be identical to that published by Collin et al.(18) .

Construction of Expression Plasmids in pAGS-3 Vector

The coding region of the human cardiac clone (hHT-1) was removed using HindIII (5`-polylinker site) and HpaI(8046) cleavages, while the pAGS-3 vector (19) was cut with NotI (polylinker), filled in with the Klenow fragment of DNA polymerase I to produce blunt ends, and then cut with HindIII (polylinker). The fragments were ligated, and the construct was verified by restriction analysis. The skeletal muscle clone as well as the H clone were transferred to pAGS-3 using HindIII-NotI sites. The resultant expression plasmids were verified by restriction mapping.

Transient Expression and Electrophysiological Recordings

For the transient expression of the hHT , and H subunits, cDNA expression plasmids were transfected by the Ca-phosphate method (20) into HEK293 cells in a molar ratio of 1:2:3, respectively. Whole cell recordings were conducted at room temperature (20-22 °C) using standard techniques(21) . Electrophysiological recordings were done 24-72 h after transfection of the HEK 293 cells. The patch electrodes were filled with an internal solution containing (in mM) 130 CsCl, 4 ATP, 5 MgCl, 5 EGTA, l0 HEPES/CsOH (pH 7.4). In bathing solution, composed of (in mM) 40 BaCl or 40 CaCl as indicated, 90 tetraethylammonium chloride, 10 HEPES (pH adjusted with KOH to 7.4), no inward directed currents were detected in nontransfected cells or in cells transfected with and subunits. The expressed currents were identified as L-type calcium channel currents due to their sensitivity to 1,4-dihydropyridine-type calcium antagonists and agonists. The inactivation kinetics were fitted with the following equation: I = I +Iexp - t/. Statistical significance was analyzed using the Student's t test (p < 0.05). Data are expressed as mean ± S.E. All chemicals were purchased from Sigma.


RESULTS

Proteases Increase Calcium Channel Current in HEK Cells Coexpressing hHT , , and Subunits

In native cardiac cells, intracellular application of trypsin caused a 3-4-fold increase in calcium channel current(13) . We obtained similar results in HEK cells in which hHT , , and were coexpressed. Intracellular perfusion with 1 mg/ml trypsin slowly increased the peak amplitude of the barium current (peak I). After a delay of 5 min, I rose from -278 to -1105 pA (Fig. 1A). No increase was observed when heat-inactivated trypsin was perfused (n = 10), indicating that the channel modification we observed was specific to the proteolytic action of trypsin. In addition to this striking increase in peak I, the inactivation time constant of I decreased during this time period from 161 to 104 ms (Fig. 1B).


Figure 1: Intracellular application of trypsin increases I in HEK cells coexpressing hHT , , and . A, time course of trypsin action. The maximum inward current (peak I) is plotted versus time. The cell was depolarized every 60 s from -80 to 20 mV. wt, wild type. B, superposition of two current traces recorded 1 min after establishing the whole cell configuration (control) and 10 min later (trypsin). C, voltage dependence of the calcium channel current. Peak I was plotted against the potential of different test depolarizations 1 min after starting to perfuse the cell with trypsin (control, opencircles) and 15 min later (trypsin, closed circles). The holding potential was -80 mV. D, effect of 1 mg/ml of the exopeptidase, carboxypeptidase A, on I elicited by a 400-ms-long depolarization from -80 to 20 mV. The inactivation time course of the currents was fitted by a single exponential with the indicated time constants.



To study the effects of trypsin on voltage-dependent activation of the calcium channel, we constructed current-voltage relationships shortly after establishing the whole cell configuration and again 10 min later. Under control conditions, the peak I had a threshold at approximately -20 mV and reached maximum at 20 mV. In the presence of trypsin, no significant shifts in the threshold or in the potential of maximum inward current were observed (Fig. 1C). The half-potential of activation was 4.1 mV 1 min after establishing the whole cell configuration and 3.2 mV 9 min later. These observations show that the region of the calcium channel complex that is responsible for voltage-dependent activation is not modulated by the proteolytic action of trypsin.

Trypsin cleaves uniformly at arginine and lysine residues(22) . To focus on the carboxyl-terminal region, we employed carboxypeptidase A, which is an exopeptidase that catalyzes the removal of amino acid residues sequentially from the carboxyl terminus (Fig. 1D). Cell dialysis with 1 mg/ml carboxypeptidase A increased peak I within 12 min from -423 to -1076 pA (the average increase was 2.41 ± 0.5 (n = 4)) and accelerated the inactivation time course of the expressed current. The results are compatible with the hypothesis that both the enhancement of the calcium channel current as well as acceleration of the inactivation time course are caused by proteolysis of the carboxyl terminus of one or more of the calcium channel subunits.

The Calcium Channel Current Stimulation by Trypsin Is Due to a Modification of the Subunit

In the next set of experiments, we addressed the question which subunit(s) of the calcium channel complex are involved in the proteolytic enhancement of I. Two currents are shown on the leftpanel of Fig. 2A, elicited by coexpression of hHT and subunits and recorded 1 and 11 min after establishing the whole cell configuration. Intracellular dialysis with trypsin increased I from -59 to -286 pA and accelerated the inactivation time course of the expressed current from 510 to 305 ms. In five cells, the average stimulation of peak I was 2.98 ± 0.51. This value is not statistically different from the increase of peak I in cells coexpressing all three subunits (, , and ). No modification of the voltage dependence of peak I by the proteolytic treatment was observed (Fig. 2B, left). These experiments provide evidence that expression of a subunit is not a prerequisite for proteolytically induced calcium channel stimulation. In cells transfected only with the subunit, within 10 min trypsin increased peak I from -49 to -135 pA and decreased the inactivation time constant from 470 to 261 ms (Fig. 2A, right). On average, trypsin increased I, within 8-15 min, by a factor of 3.37 ± 0.35 (n = 6). When we compared the current-voltage relationship before and after trypsin treatment, no appreciable difference in the threshold or in the potential of the maximum inward current was observed (Fig. 2B, right). The results show that proteolytic modification of the subunit is solely responsible for the stimulation of the calcium channel current and the shortening of inactivation time.


Figure 2: Importance of the calcium channel subunits for the proteolytic calcium channel stimulation. A, superposition of current traces elicited by 400-ms depolarizations from -80 to 20 mV 1 min after establishing the whole cell configuration (control) and 10 min later (trypsin). B, the corresponding current voltage relationships. C, superposition of current records obtained 1 min after establishing the whole cell configuration (control) and 12 min later. The experiment was done with 40 mM Ca as the charge carrier and in the presence of 1 µM (+)S202-791. The inactivation time constants are indicated. wt, wild type.



To examine whether the current stimulation depends on the charge carrier used, we performed experiments with 40 mM Ca instead of 40 mM Ba (Fig. 2C). Since the calcium current (I) induced by expression of the subunit alone was very small, we augmented I (-18 pA) by adding the calcium agonist (+)S202-791()(1 µM) to the bath solution. Within 12 min after establishing the whole cell configuration, intracellular application of trypsin increased peak I from -196 to -686 pA and accelerated the inactivation time course of I (Fig. 2C). 11 min later, the current still had a peak amplitude of -643 pA and inactivated with a time constant of 242 ms. Remarkably, even after this long period of trypsin perfusion, no slowing of the inactivation time course was observed, as has been reported for trypsin dialysis of cardiac myocytes(12) . Our observation that proteolytic treatment of HEK cells did not decelerate the inactivation time course of the expressed I is further confirmed by the results obtained in HEK cells expressing all three calcium channel subunits where peak I was increased by a factor of 3.7 ± 1.4 (n = 3) without any slowing of the inactivation time course. From these results, we conclude that the distal portion of the carboxyl-terminal tail contributes significantly to voltage-dependent but not to calcium-dependent inactivation.

Trypsin Removes Part of the Carboxyl-terminal Tail of the Subunit

A comparison of typical Is induced by coexpression of wild type , , and subunits before (left) and during (middle) trypsin perfusion and an I induced by coexpression of and subunits with a deletion mutant subunit (right), where the carboxyl terminus was removed up to amino acid 1673 (1673 mutant), is shown in Fig. 3A. In HEK cells coexpressing the wild type , intracellular perfusion of trypsin increased peak I within 10 min from -344 to -1329 pA and accelerated inactivation, and the time constant decreased from 185 to 124 ms. The shape and amplitude of the trypsin-enhanced I are very similar to the same parameters of the current induced by coexpression of the mutant 1673. The peak amplitude reached a level of -1305 pA and decayed with a time constant of 117 ms. The current densities and inactivation time courses of the trypsin-stimulated current were (using the wild type ), on average, very similar to those obtained by expression of the mutant 1673 without trypsin treatment (Fig. 3B), suggesting that trypsin, by removing a specific segment of the carboxyl-terminal tail of the wild type subunit, increases the amplitude of the calcium channel current. This is further substantiated by an experiment in which we perfused a HEK cell coexpressing l673, , and with the same concentration of trypsin that caused a 3-fold increase of peak I in cells expressing wild type subunit alone. Even after 15 min of perfusion, no increase of peak I was detected (Fig. 3C). The current had a similar shape 1 min and 15 min after establishing the whole cell configuration (Fig. 3C, inset). No increase of the calcium channel current was detectable in any of the eight cells tested. This reinforces that the part of the carboxyl-terminal tail of the subunit that is digested by proteolytic treatment with trypsin or carboxypeptidase A was removed by mutagenesis and is responsible for the proteolytically induced effects observed. In a few experiments, carboxypeptidase A was tested and acted in the same way as trypsin (data not shown). External application of trypsin or carboxypeptidase A was without effect (data not shown). It is of interest that in a recent report (23) currents elicited by the coexpression of carboxyl-terminal deletion mutants of cardiac in Xenopus oocytes were 4-6-fold larger than those of the wild type subunit.


Figure 3: Lack of proteolytic I stimulation in HEK cells transfected with a deletion mutant of the subunit carboxyl terminus. A, representative current traces obtained by a 400-ms-long depolarization from -80 to 20 mV induced by expression of the indicated subunit combination. B, statistical evaluation of current densities and inactivation time constants of I directed by expression of the indicated subunit combinations. The test potential was 20 mV. Data represent mean ± S.E. n = 5-7. C, time course of the effect of trypsin perfusion on a HEK cell coexpressing 1673, , and subunits. wt, wild type.




DISCUSSION

Intracellular perfusion of HEK cells coexpressing wild type , , and subunits with trypsin increased I to an extent similar to that reported for trypsin stimulation of class C calcium channel currents in cardiac myocytes, vascular smooth muscle cells, and the A7r5 cell line(4, 13, 24) . In all three cell types, intracellularly applied trypsin enhanced the amplitude of the calcium channel current 3-4-fold without changing its voltage dependence. Our results demonstrate that proteolytic stimulation of cardiac L-type calcium currents is due to digestion of cytoplasmic regions of the carboxyl terminus of the subunit. Since no enhancement of peak I was observed in HEK cells expressing the carboxyl-terminal deletion mutant 1673, it is obvious that digestion of amino acids proximal to amino acid 1673 does not result in any further increase of the calcium channel current.

In cardiac myocytes, a 10-fold slowing of the inactivation time course of the calcium current was observed exclusively with Ca as the charge carrier(13) , a phenomenon which we never observed in our experiments. In this respect, the recombinant cardiac calcium channels expressed in HEK cells resemble more the ``smooth muscle type'' than the ``cardiac muscle type'' calcium channel. In smooth muscle cells, no significant effect of trypsin treatment on the inactivation time course of I has been reported(4, 24) . Using Ba as the charge carrier, we observed an acceleration in the inactivation time course of the expressed currents upon intracellular application of trypsin, similarly to that reported for the native calcium channel(13) . From these results, we suggest that the carboxyl terminus must be an important constituent of the calcium channel protein that is intimately involved in the voltage-dependent inactivation process. It might be argued that trypsin affects other structures of the calcium channel protein that are involved in the inactivation process(30) . However, the equally fast inactivation of Is induced by coexpression of the mutant 1673 subunit and the lack of any trypsin effect in this mutant clearly show that the removal of part of the carboxyl terminus is responsible for the faster inactivation time course of I. This does not exclude other regions of regulating inactivation such as IS and flanking regions(30) . One possibility is that folding of the distal region of the carboxyl terminus of the subunit may influence the voltage sensor-induced changes in motif IS and flanking regions(30) . Several lines of experimental evidence indicate that distinct portions of skeletal, cardiac, and neuronal type calcium channel subunits influence the channel kinetics(28, 29, 30) , although the involvement of the carboxyl terminus has not yet been suggested. The similar increase in current density and acceleration of inactivation either with Ba or Ca as charge carrier indicates the possibility that the E-F hand (31) on the carboxyl-terminal tail may not play a critical role in proteolytic modulation of channel function(32) .

These observations shed new light on the molecular nature of the inactivation of calcium channels. Thus far, certain constituents of the multiple inactivation machineries were identified for K, Na, and Ca channels (8, 9, 11, 12, 25, 30, 32, 33, 36). It has been shown that removal, replacement, or mutagenesis of these segments slows or eliminates certain inactivation processes. Calcium channels carry several segments that can influence kinetic properties. The carboxyl-terminal region of calcium channels is unique in that it is also involved in regulating the availability of the channel for pore opening, resulting in increased open state probability(23) . Whether these two groups of phenomena are just two aspects of the same molecular mechanism or whether they can be described by two independent mechanisms is open for further experimentation.

In native cardiac myocytes, it has been shown that the effects of intracellular trypsin application and stimulation of the -adrenergic receptor by isoproterenol are not additive but saturable. This leads to speculation that trypsin, in a manner similar to -adrenergic-induced phosphorylation, removes the blocking action of part of the unphosphorylated subunit(13) . Unfortunately, it is very difficult to obtain direct evidence for protein kinase A-dependent phosphorylation of recombinant calcium channels (2, 3) . Since the deleted part of the 1673 mutant subunit encompasses only one consensus phosphorylation site, i.e. Ser, it is tempting to speculate that this serine is phosphorylated by protein kinase A, as has been proposed earlier(5, 34) .

Our finding that trypsin increased I elicited by expression of the subunit alone may be viewed as an apparent contradiction to our previous experiments using the skeletal muscle subunit () stably transfected into mouse L-cells without and with a subunit. Intracellular trypsin treatment of cells coexpressing and amplified I by 3-4-fold, accompanied by a slowing of the current kinetics. In the absence of a subunit, the was unaffected by trypsin or carboxypeptidase A(25) . The observations for the skeletal muscle isoform are clearly consistent with the notion that proteolytic digestion abolishes the - interaction, which does not appear to be the case at least functionally, in cardiac muscle. Recently, the - interaction site was mapped and found to be located on the intracellular connecting loop of motifs I and II of various subunit isoforms(26) . The skeletal muscle subunit has an obligatory requirement for the subunit to mimic the native state(35) , whereas cardiac alone functions similar to the native-like channel (15) . Truncation of the carboxyl terminus of the skeletal subunit does not appear to influence the expressed current(27) , an observation clearly opposite to the present data in which a region on the carboxyl-terminal tail of the cardiac subunit has a significant influence on channel kinetics. The corresponding segments on skeletal and cardiac subunits clearly define distinct functional domains. We feel, therefore, that our present results provide definitive evidence for an important qualitative difference in the regulation of cardiac and skeletal L-type voltage-dependent calcium channels. Recognition of the role of the carboxyl terminus in the inactivation process illustrates the multiple ways in which nature provides different but perhaps related structures (30, 32, 36) in regulation of voltage-gated channels.


FOOTNOTES

*
This work was supported by grants from the Deutsche Forschungsgemeinschaft (UK), by National Institutes of Health Grants P01 HL 22619-17, R37HL 43231-06, and PO1 HL 41496-06, and by the Tanabe Seiyaku Fund for Molecular Biophysics. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
A Research Associate in the Program of Excellence (HL-41496).

On leave of absence from Loránd Eötvös University, Budapest Hungary. To whom correspondence and reprint requests should be addressed: Institute of Molecular Pharmacology and Biophysics, University of Cincinnati, College of Medicine, 231 Bethesda Ave., P. O. Box 670828, Cincinnati, OH 45267-0828. Tel.: 513-558-2466; Fax: 513-558-1778.

The abbreviation used is: (+)S202-791, isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-pyridine carboxylate.


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