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The amino side of the C-terminus determines fast inactivation of the T-type calcium channel _1G

Mik Staes, Karel Talavera, Norbert Klugbauer*, Jean Prenen, Lubica Lacinová*, Guy Droogmans, Franz Hofmann* and Bernd Nilius

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

1. We analysed the kinetic properties of the fast inactivating T-type calcium channel _1G in HEK 293 cells transfected with different _1G chimeras, containing the N-terminus, III-IV linker or various C-terminal regions of the slowly inactivating L-type _1C.

2. A highly negatively charged region of 23 amino acids at the amino side of the intracellular carboxy terminus of _1G was found to be critical for fast inactivation.

3. The N-terminus of _1G does not seem to be necessary for inactivation of the T-type calcium channel because replacement of the _1G N-terminus with the _1C N-terminus did not influence channel kinetics at all.

4. Replacing the III-IV linker of _1G with that of _1C decreased the rate of inactivation at -20 mV from 15.8 ± 1.8 to 8.5 ± 1.1 ms, and shifted the potential for half-maximal inactivation from -69.6 ± 0.8 to -54.0 ± 1.7 mV. However, these parameters were not significantly different at other potentials.

5. We suggest a putative 'ball-and-chain'-like mechanism for inactivation in which the negative charges function as an acceptor domain for a ball, hypothetically located at a different intracellular part of the channel. In addition, transferring the IQ motif and EF hand of _1C to _1G does not confer Ca²⁺-dependent inactivation on _1G, suggesting that other sequences besides the C-terminus are needed for Ca²⁺-dependent inactivation of _1C.

	INTRODUCTION

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Voltage-dependent Ca²⁺ channels (or voltage-operated Ca²⁺ channels, VOCCs) cause a rapid influx of Ca²⁺ into cells upon membrane depolarisation. They play an important role in neurons, and skeletal, cardiac and smooth muscle, but also in non-excitable cells like endocrine cells, lymphocytes and spermatocytes. These channels are involved in synaptic signal transduction, excitation-contraction coupling, excitation-secretion coupling, pacemaker function, cell growth, cell proliferation and apoptosis (Cohen et al. 1988; Tsien et al. 1991; Hofmann et al. 1994; Zhang et al. 1994).

Based on their electrophysiological and pharmacological properties, VOCCs can be functionally classified into different subtypes (L-, N-, P/Q-, R- and T-type) (Tsien et al. 1991; Zhang et al. 1993; Hofmann et al. 1999). T-type calcium channels show some unique biophysical properties, which are absent in other VOCCs. They activate at much more negative potentials, ranging from -50 to -30 mV with a peak current around -20 mV (Droogmans & Nilius, 1989; Chen & Hess, 1990; Huguenard, 1996) and are therefore also called low voltage-activated (LVA) channels. Inactivation of the macroscopic current is voltage dependent but Ca²⁺ independent, resulting in a typical criss-crossing current pattern (Droogmans & Nilius, 1989; Chen & Hess, 1990). T-type channels also deactivate slowly, giving rise to long-lasting tail currents during repolarisation (Droogmans & Nilius, 1989; Chen & Hess, 1990). These properties are functionally important for induction of large inward Ca²⁺ currents following short depolarisations during an action potential (Nilius et al. 1985; Huguenard, 1996). T-type channels may play a critical role in the generation of electrophysiological rhythms, such as the cardiac pacemaking of sino-atrial node cells (Nilius, 1986; Tseng & Boyden, 1989; Lei et al. 1996) and are part of an internal clockwork system controlling oscillations in thalamocortical networks (Destexhe et al. 1993; Tsien, 1998). They are also involved in excitation-induced secretion processes (Barrett et al. 1996; Chen et al. 1999). The subunit composition of LVA channels is still unknown, but they contain at least one ₁ subunit, responsible for voltage sensing, ion selectivity, ion conduction and binding of some channel blockers (Hofmann et al. 1999). Three different ₁ subunits of T-type channels have been identified and cloned: _1G, _1H and _1I (Perez-Reyes et al. 1998; Cribbs et al. 1998; Klugbauer et al. 1999; Lee et al. 1999). The _1G subunit of the T-type calcium channel contains four homologous domains (repeats I to IV) each of them consisting of six transmembrane -helices (S1 to S6). Interactions between _1G co-expressed with an ₂ and/or subunit have not (Lacinova et al. 1999) or have (Dolphin et al. 1999) been observed.

At present, the structure-function relationship for the gating mechanism of T-type calcium channels is not known. To investigate the fast inactivation mechanism of T-type channels, we have created different chimeras by replacing parts of the T-type _1G protein (Klugbauer et al. 1999) with the corresponding sequences of the slowly inactivating L-type _1Cb Ca²⁺ channel (Biel et al. 1990). Inactivation of the L-type Ca²⁺ channel is also Ca²⁺ dependent, a process that is controlled by intracellular sequences in the carboxy terminus, which have been identified as calmodulin (IQ motif) and calcium (EF hand) binding sites (Wei et al. 1994; de Leon et al. 1995; Qin et al. 1999; Zühlke et al. 1999). We have therefore also constructed different C-terminal chimeras containing either or both of the IQ and EF motifs to investigate whether Ca²⁺ dependency of inactivation can be transferred from _1C to _1G.

The voltage-gated Na⁺ channel is, like the T-type Ca²⁺ channel, composed of four homologous internal repeats, each of them consisting of six putative transmembrane segments (Noda et al. 1984). Since the hydrophobic IFM motif in the intracellular linker between region III and IV is involved in the fast inactivation of voltage-dependent sodium channels (Stühmer et al. 1989; Patton et al. 1993; Catterall, 1999), we have also removed this region from _1G and replaced it with the corresponding part of _1C in order to investigate if the fast inactivation of _1G is also due to specific apolar residues in the III-IV linker.

The intracellular amino terminus of K⁺ channels is involved in their inactivation mechanism based on a ball-and-chain interaction (Armstrong & Bezanilla, 1977; Hoshi et al. 1990; Zagotta & Aldrich, 1990). We have therefore replaced this terminus of _1G with that of _1C with the purpose of investigating whether the T-type N-terminus is necessary for inactivation.

	METHODS

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Construction of calcium channel chimeras

Chimeras were obtained using the standard PCR overlap extension technique with the mouse _1G cDNA, pc3LVA1, accession number AJ012569 (Klugbauer et al. 1999) and the rabbit _1Cb cDNA, accession number X55763 (Biel et al. 1990) as the template DNA. If necessary, subfragments of _1G or _1Cb were first subcloned into pUC18 (New England Biolabs) or pBluescript II KS+ (Stratagene) as follows. GCN: Hind III _1G fragment (1292 bp) in pUC18 (Subclone 1); GCL: Hind III-Acc65 I _1G fragment (4539 bp) in pUC18 (Subclone 2); GCC₁-GCC₄: EcoR I-Xho I _1Cb fragment (4496 bp) in pBluescript II KS+ (Subclone 3); GCC₅: Xho I _1G fragment (1767 bp) in pBluescript II KS+ (Subclone 4).

A part of the insert was replaced by the corresponding sequence of the _1Cb cDNA. Replacement sequences were created by standard PCR overlap extension. The primers and template DNA for each chimera are given below (Table 1).

The sequence of each chimera was verified by sequence analysis. The exact locations of sequence exchanges between _1G and _1Cb are shown in Fig. 1.

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Figure 1 Schematic representation of 1G-1C chimeras Black boxes indicate intracellular parts from 1G. Open boxes represent fragments of 1C. The exact locations of the exchanges between 1G and 1C are given by the numbers of the corresponding amino acids (AA).

Cell culture and transfection of HEK 293 cells

Human embryonic kidney cells (HEK 293 cell line) were grown in Dulbecco's modified Eagle's medium containing 10% (v/v) human serum, 2 mM L-glutamine, 2 U ml^-1 penicillin and 2 mg ml^-1 streptomycin at 37°C in a humidity controlled incubator with 10% CO₂. The cells were transiently transfected with the expression vectors using LipofectAMINE PLUS reagent according to the instructions of the manufacturer (Gibco-BRL Life Technologies). Expression vector pc3LVA1 was used for expression of the wild-type (WT) _1G channel in HEK 293 cells.

Solutions

The standard extracellular solution was Krebs solution, containing (mM): 150 NaCl, 6 KCl, 1 MgCl₂, 1.5 CaCl₂, 10 glucose, 10 Hepes, titrated to pH 7.4 with 1 M NaOH, with an osmolarity of 320 ± 5 mosmol l^-1. While measuring calcium currents, the cells were perfused with a bath solution containing (mM): 130 N-methyl-D-glucamine, 20 CaCl₂ or BaCl₂, 5 CsCl, 10 Hepes, 1 MgCl₂, 5 glucose, titrated to pH 7.4 with 1 M HCl, with an osmolarity of 320 ± 5 mosmol l^-1. The pipettes were filled with a solution containing (mM): 102 CsCl, 5 Hepes, 5 MgCl₂, 5 Na₂-ATP, 10 TEA-Cl, 10 EGTA, titrated to pH 7.4 with 1 M CsOH, with an osmolarity of 270 ± 5 mosmol l^-1.

Electrophysiology

Currents were recorded in the whole-cell configuration of the patch-clamp technique using an EPC-7 (List Electronics, Darmstadt, Germany) or EPC-9 (HEKA Electronics, Lambrecht/Pfalz, Germany) patch-clamp amplifier and filtered with an eight-pole Bessel filter (Kemo, Bekenham, UK). For control of voltage-clamp protocols and data acquisition, we used pCLAMP 6 software (Axon Instruments, Foster City, CA, USA) run on an IBM-compatible PC connected to the amplifier via a TL-1 DMA interface (Axon Instruments). Rapid exchange of bath solution occurred via a multi-barrelled pipette connected to solution reservoirs and was controlled by a set of magnetic valves. Patch electrodes were pulled from Vitrex capillary tubes (Modulohm, Herlev, Denmark) using a DMZ-Universal puller (Zeitz-instruments, Augsburg, Germany). When filled with pipette solution they had a DC resistance between 2 and 5 M. An Ag-AgCl wire was used as the reference electrode. The cell capacitance and series resistance were electronically compensated. Current traces were filtered at 2 kHz and digitised at 5-10 kHz. The experiments were performed at room temperature (20-25°C).

Data analysis

Electrophysiological data were analysed and fitted using the WinASCD software package (G. Droogmans, Laboratory of Physiology, KU Leuven). Peak I-V relations were fitted to:

I = ( g_max(V - V_rev) ) / ( 1 + exp[-(V - V_act)/s_act] )

where I is the measured current (in pA pF^-1), g_max the slope conductance (in nS pF^-1), V the test potential, V_rev the reversal potential, V_act the potential of half-maximal activation (all in mV) and s_act (in mV) the slope parameter for activation.

Steady-state inactivation (h ) was determined from the peak current during an 80 ms test pulse to -20 mV following a long-lasting pre-pulse (5120 ms) to various potentials from -100 to -25 mV in steps of 5 mV. This protocol was repeated every 2 s from a holding potential of -100 mV. These peak currents were normalised to that following a pre-pulse to -100 mV, i.e.:

h = I_peak(V_pre)/I_peak(-100).

Current traces were first corrected for linear background currents. The voltage dependence of this parameter was fitted to the Boltzmann equation:

h (V) = 1/{1 + exp[(V - V_inact)/s_inact]}, (2)

where V_inact represents the potential for half-maximal inactivation and s_inact is a slope factor.

For statistical analysis and graphical presentation of the data we used Origin version 5.0. Pooled data are given as means ± S.E.M. of all the measurements. Results were tested for significance using Student's unpaired t test with P < 0.05 and P < 0.01 as the level of significance. To account for variations in cell size, we have expressed the current amplitudes per unit of membrane capacitance in Fig. 7A.

	RESULTS

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Molecular structure of _1G-_1C chimeras

Figure 1 gives a shematic representation of all discussed chimeras, in which a fragment of the _1G channel has been replaced by the corresponding part of _1C. In GCN, the 80 amino acids in the amino terminal part of _1G have been replaced with 124 amino acids from the N-terminus of _1C. Amino acids 1542 to 1618 from _1G have been replaced with amino acids 1195 to 1243 from _1C in chimera GCL. GCC₁ is composed of the _1G channel in which the C-terminal amino acids 1863 to 2295 have been replaced with those of _1C from 1505 to 2166. In the C-terminal chimeras GCC₂, GCC₃ and GCC₄, fewer amino acids have been replaced compared to GCC₁. For GCC₂ amino acids 2024 to 2295 from _1G have been replaced by those from 1690 to 2166 in _1C, while in GCC₃ only the distal 126 amino acids at the C-terminus have been replaced with amino acids 1929 to 2166 from _1C. In GCC₄, only the first 23 amino acids of the C-terminus have been conserved while the following distal part of _1G has been replaced with amino acids 1540 to 2166 from _1C. The C-terminus of chimera GCC₅ is exactly the opposite of the C-terminus of GCC₂: the first 161 amino acids have been replaced by amino acids 1505 to 1689 from _1C.

Activation properties of _1C and _1G

Figure 2A shows representative current traces for _1C and _1G expressed in HEK cells during voltage steps between -90 and 60 mV from a holding potential of -100 mV using either 20 mM Ca²⁺ (left) or 20 mM Ba²⁺ (right) as the charge carrier. Inactivation of L-type currents was much slower if the current was carried by Ba²⁺ (Fig. 2A, top). In contrast, inactivation of WT _1G was voltage dependent, but did not depend on the charge carrier, illustrating the Ca²⁺-independent inactivation of T-type calcium currents (Fig. 2A, bottom). Figure 2B shows the normalised I-V relations for _1C and _1G for both Ca²⁺ and Ba²⁺ as a charge carrier. Curves for _1C were shifted by about 50 mV to more positive potentials, consistent with the 'high threshold' nature of this channel as compared with the 'low threshold' _1G.

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Figure 2 Comparison of T- and L-type currents in 20 mM Ca2+ and 20 mM Ba2+ A, representative activation current traces for 1C (top) and 1G (bottom), in 20 mM Ca2+ (left) or 20 mM Ba2+ (right). The currents result from an activation step protocol that consists of subsequent 160 ms test pulses from -90 to 60 mV in steps of 10 mV, starting from a holding potential of -100 mV. Only traces representing visible currents are shown. The time interval between different episodes was 2 s. B, corresponding normalised current-voltage relationships for 1C and 1G for 20 mM Ca2+ (left) or 20 mM Ba2+ (right).

Involvement of III-IV linker in inactivation of _1G

Figure 3A shows representative current traces for _1G and the III-IV linker chimera GCL using 20 mM Ca²⁺ as the charge carrier. These traces were recorded during voltage steps between -80 and 30 mV (_1G) or -80 and 50 mV (GCL) applied from a holding potential of -100 mV. Inactivation time constants, _inact, obtained from a single exponential fit to the decaying phase of the current, are shown as a function of voltage in Fig. 3B. It is obvious that substitution of the III-IV linker region has only minor effects on the rate of inactivation. Steady-state inactivation parameters for WT _1G and GCL, as obtained from a fit of the voltage dependence of h (not shown) to eqn (2), are summarized in Fig. 6A and B together with _inact for a test pulse at -20 mV (Fig. 6C). V_inact and _inact of GCL (V_inact = -54.0 ± 1.7 mV; _inact = 8.5 ± 1.1 ms) were significantly decreased compared to _1G (V_inact = -69.6 ± 0.8 mV; s_inact = 4.2 ± 0.3 mV; _inact = 15.8 ± 1.8 ms), while s_inact of GCL (s_inact = 4.4 ± 0.3 mV) was unaffected.

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Figure 3 Activation currents and inactivation time constants of 1G and GCL A, representative activation current traces in 20 mM Ca2+ for 1G (left) and GCL (right). The currents resulted from an activation step protocol that consists of subsequent 160 ms test pulses from -90 to 60 mV in steps of 10 mV, starting from a holding potential of -100 mV. Only traces representing visible currents are shown. The time interval between different episodes was 2 s. B, time constants of inactivation (inact) at different test potentials. Statistical analysis (Student's unpaired t test) was applied to the resulting data from at least 6 different measurements.

The amino terminus of _1G does not influence kinetics of inactivation

As shown at the bottom of Fig. 6, the inactivation properties of the amino terminal chimera GCN (V_inact = -66.0 ± 0.6 mV; s_inact = 3.9 ± 0.2 mV; _inact = 13.6 ± 0.8 ms) were not different from those of _1G and will not be discussed further.

The carboxy terminus is essential for fast inactivation of _1G

Figure 4 compares current traces with 20 mM Ca²⁺ as the charge carrier in response to voltage steps between -80 and 30 mV applied from a holding potential of -100 mV for _1G and the C-terminal chimeras GCC₁, GCC₄ and GCC_5. Compared to WT _1G, inactivation was clearly slower in GCC₁ and GCC₅, containing a replacement of the complete C-terminus or only the first 161 C-terminal amino acids, respectively, while no difference was observed for GCC₄. The time constants of inactivation, _inact, plotted in Fig. 4B, were 2 to 3 times larger for GCC₁ and GCC₅ than the corresponding values of WT _1G, whereas those for GCC₄ were not significantly different.

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Figure 4 Activation currents and inactivation time constants of 1G and C-terminal chimeras A, representative activation current traces in 20 mM Ca2+ for 1G (top left), GCC1 (top right), GCC4 (bottom left) and GCC5 (bottom right). The currents resulted from an activation step protocol that consists of subsequent 160 ms test pulses from -90 to 60 mV in steps of 10 mV, starting from a holding potential of -100 mV. Only traces representing visible currents are shown. The time interval between different episodes was 2 s. B, time constants of inactivation (inact) at different test potentials. Statistical analysis (Student's unpaired t test) was applied to the resulting data from at least 6 different measurements.

Figure 5 shows representative current traces from _1G, GCC₁, GCC₄ and GCC₅ with 20 mM Ca²⁺ as the charge carrier in response to a depolarising voltage step to -20 mV following a 5120 ms pre-pulse to potentials ranging from -100 to -25 mV in increments of 5 mV. The time courses of inactivation for GCC₁ and GCC₅ were again much slower compared to that of _1G. Steady-state inactivation curves, obtained from the peak current amplitudes, are depicted in Fig. 5B. Compared to _1G (V_inact = -69.6 ± 0.8 mV; s_inact = 4.2 ± 0.3 mV), the curve for GCC₄ (V_inact = -56.7 ± 1.0 mV; s_inact = 4.5 ± 0.1 mV) was shifted 13 mV to the right, while the slope of the curve was unaffected. In contrast, for GCC₁ (V_inact = -66.9 ± 1.2 mV; s_inact = 5.8 ± 0.3 mV) and GCC₅ (V_inact = -66.5 ± 1.5 mV; s_inact = 6.2 ± 0.4 mV), the slope factors were increased, while no significant shift occurred.

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Figure 5 Inactivation currents and steady-state inactivation curves of 1G and C-terminal chimeras A, representative inactivation current traces for 1G, GCC1, GCC4 and GCC5. The currents resulted from a protocol that consists of an 80 ms test pulse at -20 mV, following variable 5120 ms pre-pulses from -100 to -25 mV in steps of 5 mV. The holding potential was -100 mV. The interval between different episodes was 2 s. B, the average steady-state inactivation curves.

The average time constants of inactivation and the parameters of steady-state inactivation of the five different C-terminal chimeras are depicted in Fig. 6. V_inact was significantly shifted to less negative potentials for GCC₂ (-51.5 ± 1.3 mV), GCC₃ (-60.1 ± 2.1 mV) and GCC₄ (-56.7 ± 1.0 mV) compared to WT _1G (-69.6 ± 0.8 mV) (P < 0.01), whereas s_inact for GCC₁ (5.8 ± 0.3 mV) and GCC₅ (6.2 ± 0.4 mV) was significantly different from that of the WT channel (4.2 ± 0.3) (P < 0.01). Chimeras GCC₁ (_inact = 43.6 ± 2.9 ms) and GCC₅ (_inact = 30.5 ± 1.9 ms) inactivated significantly slower than _1G (_inact = 15.8 ± 1.8 ms) (P < 0.01) as already shown in Fig. 5.

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Figure 6 Kinetic characteristics of inactivation of different T-channel chimeras A, potentials of half-maximal steady-state current inactivation (Vinact). B, slopes of the mean inactivation curves (sinact). C, time constants for inactivation at a test pulse of -20 mV (inact). Columns and corresponding error bars represent mean and S.E.M. values, respectively. Statistical analysis (Student's unpaired t test) was applied to the resulting data from at least 5 different measurements. Significant differences compared to WT 1G: *P < 0.05, **P < 0.01.

Calcium-dependent inactivation cannot be transferred from _1C to _1G

Inactivation properties of wild-type _1G in 20 mM Ba²⁺ and 20 mM Ca²⁺ are compared in Fig. 7. Figure 7A represents the peak I-V relation curves showing a small shift to more positive potentials for Ba²⁺. Current amplitudes in Ba²⁺ were smaller than Ca²⁺ currents. Time constants of inactivation (Fig. 7B) were the same in Ba²⁺ and Ca²⁺, except at positive test potentials where inactivation was slightly faster in Ba²⁺ than in Ca²⁺. Steady-state inactivation curves, as depicted in Fig. 7D, were obtained from peak current amplitudes during steps to -20 mV following long-lasting steps to various potentials, an example of which is shown in Fig. 7C for Ca²⁺ and Ba²⁺. The inactivation curve for Ba²⁺ (V_inact = -57.4 ± 1.0 mV; s_inact = 4.9 ± 0.4 mV; _inact = 14.1 ± 1.6 ms) was shifted 12 mV to the right compared to Ca²⁺ (V_inact = -69.6 ± 0.8 mV; s_inact = 4.2 ± 0.3 mV; _inact = 15.8 ± 1.8 ms), which is consistent with the observed shift of the peak I-V relations shown in Fig. 7A.

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Figure 7 Inactivation properties of 1G in Ca2+ compared to Ba2+ A, current-voltage relationships of 1G in 20 mM Ba2+ () and 20 mM Ca2+ (). B, time constants of inactivation (inact) at different test potentials in Ca2+ and Ba2+. Statistical analysis (Student's unpaired t test) was applied to the resulting data from at least 6 different measurements. C, representative inactivation current traces in Ca2+ and Ba2+ for 1G. The currents resulted from a protocol that consists of an 80 ms test pulse at -20 mV, following variable 5120 ms pre-pulses from -100 to -25 mV in steps of 5 mV. The holding potential was -100 mV. The interval between different episodes was 2 s. D, the average steady-state inactivation curves of 1G in Ba2+ () and Ca2+ (). Statistical analysis (Student's unpaired t test) was applied to the resulting data from at least 6 different measurements.

Figure 8 compares the slopes of the steady-state inactivation curves, s_inact, and time constants of inactivation at -20 mV, _inact, for WT _1G and all C-terminal chimeras. Mean values for both Ba²⁺ and Ca²⁺ are compared to the mean value of _1G for Ca²⁺ (*). The mean of each chimera for Ba²⁺ is also compared to the mean of _1G for Ba²⁺ (+). For _1G and each chimera separately, a comparison of the mean values for Ba²⁺ and the mean for Ca²⁺ is shown (). The presence or absence of the IQ motif and EF hand is indicated on the left. s_inact for GCC₁ (+IQ/+EF) and GCC₅ (+IQ/+EF) in the presence of either Ba²⁺ or Ca²⁺ was significantly higher than for _1G in the presence of Ca²⁺, whereas GCC₄ (+IQ/-EF) only differed from _1G if Ba²⁺ was the charge carrier. The slope parameter of GCC₅ (+IQ/+EF) was significantly larger than that of _1G in the presence of Ba²⁺. GCC₂ (-IQ/-EF) and GCC₄ (+IQ/-EF) were the only chimeras that showed a significantly different increase in the slope of steady-state inactivation between Ca²⁺ and Ba²⁺.

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Figure 8 Comparison of sinact and inact in Ca2+ and Ba2+ for 1G and C-terminal chimeras The figure shows the slopes of the steady-state inactivation curves, sinact, and the time constants of inactivation for a test pulse at -20 mV, inact, for WT 1G and all chimeras. The presence (+) or absence (-) of the IQ or EF motifs is shown on the left side. Asterisks (*) indicate significant differences between the means of chimeras in Ba2+ or Ca2+, and the mean of 1G in Ca2+. Crosses (+) indicate significant differences between the mean of each chimera for Ba2+ and the mean of 1G for Ba2+. For 1G and each chimera separately, a significant difference between the mean for Ba2+ and the mean for Ca2+ is shown as an open circle (). Single symbols are used for P < 0.05 and double symbols for P < 0.01.

Compared to _1G with Ca²⁺, _inact for GCC₁ (+IQ/+EF) was increased with both Ba²⁺ and Ca²⁺. Also _inact of GCC₅ (+IQ/+EF) was increased for Ca²⁺, while inactivation was slower for GCC₄ (+IQ/-EF) with Ba²⁺. Compared to _1G in Ba²⁺, GCC₁ (+IQ/+EF) and GCC₅ (+IQ/+EF) showed a slower inactivation if Ba²⁺ was the charge carrier, while GCC₄ (+IQ/-EF) was much faster. In contrast with _1G, chimeras GCC₁ (+IQ/+EF), GCC₂ (-IQ/-EF), GCC₄ (+IQ/-EF) and GCC₅ (+IQ/+EF) inactivated faster with Ba²⁺compared to Ca²⁺, while no difference was seen for GCC₃ (-IQ/-EF).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

We have studied a number of chimeras of T- and L-type Ca²⁺ channels in order to investigate the inactivation mechanism of the T-type Ca²⁺ channel _1G at the molecular level. This is important for several reasons. (1) The inactivation mechanism of T-type channels seems to be different from that of other voltage-dependent channels. It has indeed been shown that the macroscopic time constant of inactivation is closely correlated with the latency time of channel opening in single-channel recordings, suggesting that macroscopic inactivation may reflect delayed microscopic activation rather than a channel transition to an absorbing closed state (Droogmans & Nilius, 1989; Chen & Hess, 1990; Serrano et al. 1999). (2) In contrast with L-type channels, T-type calcium channels lack Ca²⁺-dependent inactivation (Bossu et al. 1985; Chen & Hess, 1990; Randall & Tsien, 1996). (3) Some channel properties, which may be related to the mechanism of inactivation, are essential for several biological functions, e.g. role in pace-making (Nilius, 1986; Droogmans & Nilius, 1989; Chen & Hess, 1990; Randall & Tsien, 1996) and Ca²⁺ entry during repolarization (Tseng & Boyden, 1989; Lei et al. 1996).

As to current inactivation, it is obvious that the substitution of the III-IV linker region (GCL) affects the inactivation properties of _1G. V_inact and _inact are changed significantly. This is not an unexpected finding, since this structure is responsible for fast inactivation of voltage-gated Na⁺ channels, which have a structure analogous to that of _1G, although the III-IV linker of _1G does not contain the IFM motif, necessary for fast inactivation of the sodium channel (Stühmer et al. 1989; Patton et al. 1993; Catterall, 1999). This indicates that the III-IV linker influences _1G inactivation via another mechanism.

On the other hand, the cytoplasmic C-terminal end seems to play a key role in inactivation, since the removal of a region of 161 amino acids at the amino side of this C-terminus slowed down inactivation for GCC₅ by a factor of 2-3. However, if only the first 23 amino acids of the C-terminus were conserved in GCC₄, the rate of inactivation was similar to _1G. This relatively small region contains 10 glutamate residues forming a negatively charged stretch adjacent to the intracellular part of the channel pore. The protein sequence of this region is also highly conserved in _1H (Cribbs et al. 1998) and _1I (Lee et al. 1999). An intriguing tentative mechanism could be a 'ball-and-chain'-type inactivation process as described for K⁺ channels (Armstrong & Bezanilla, 1977; Hoshi et al. 1990; Zagotta & Aldrich, 1990) whereby the proximal part of the C-terminus functions as a negatively charged acceptor region for a ball located in another intracellular part of the channel. An involvement of the intracellular N-terminus, as observed for K⁺ channels, can be ruled out since inactivation was not affected in GCN. The faster inactivation of GCL, in which all 77 amino acids of the III-IV linker are replaced by the shorter linker of _1C (containing only 49 residues), could be due to an improved accessibility of the C-terminal acceptor region for some attracted ball during inactivation. The shift of the potential of half-maximal steady-state current inactivation, V_inact, towards less negative potentials for GCC₂, GCC₃ and GCC₄ resulting from the replacement of respectively 272, 126 and 410 amino acids from the _1G C-terminus by the corresponding _1C region containing respectively 477, 237 and 627 amino acids may indicate that movement of the chimerical C-terminus during a putative ball-acceptor interaction requires more energy. For the slowly inactivating chimeras GCC₁ and GCC₅, this positive shift of V_inact was not observed when the C-terminal acceptor region was replaced and this may cause a lower impact of the C-terminus on the remaining inactivation process.

We were not able to transfer the calcium dependency of inactivation from _1C to _1G. Chimeras GCC₁ and GCC₅, both containing the EF hand as well as the IQ motif, even showed a slower inactivation in Ca²⁺ compared to WT _1G. In fact, inactivation was slower in Ca²⁺ than in Ba²⁺ for those chimeras. These results confirm the hypothesis that other amino acid sequences, outside the C-terminus of _1C, could be involved in Ca²⁺-dependent inactivation of _1C, like a binding region for constitutive, Ca²⁺-independent tethering of calmodulin to the channel (Peterson et al. 1999, 2000). Obviously, _1G does not have a proper site for constitutive calmodulin binding which could allow the C-terminal chimeras containing the IQ motif and EF hand (GCC₁ and GCC₅) to inactivate in a Ca²⁺-dependent way.

Further investigation including measurements at the single channel level is necessary to localise the critical domains for inactivation more precisely and to unravel the possible interactions between different regions of the _1G channel.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

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Acknowledgements

We thank Drs J. Eggermont, K. H. Suh, J. Papassotiriou and R. Vennekens for helpful discussions. The expert technical assistance of M. Crabbé, H. Van Weijenbergh and M. Schuermans is greatly acknowledged. This work was supported by the Belgian Federal Government, the Flemish Government and the Onderzoeksraad KU Leuven (GOA 99/07, F.W.O. G.0237.95, F.W.O. G.0214.99, F.W.O. G.0136.00; Interuniversity Poles of Attraction Program, Prime Ministers Office IUAP Nr.3P4/23, and C.O.F./96/22-A069), and by 'Levenslijn' (7.0021.99).

Corresponding author

B. Nilius: Laboratorium voor Fysiologie, KU Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium.

Email: bernd.nilius{at}med.kuleuven.ac.be

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