Multiple Structural Elements Contribute to the Slow Kinetics of the Cav3.3 T-type Channel*

Molecular cloning and expression studies established the existence of three T-type Ca2+ channel (Cav3) α1 subunits: Cav3.1 (α1G), Cav3.2 (α1H), and Cav3.3 (α1I). Although all three channels are low voltage-activated, they display considerable differences in their kinetics, with Cav3.1 and Cav3.2 channels activating and inactivating much faster than Cav3.3 channels. The goal of the present study was to determine the structural elements that confer the distinctively slow kinetics of Cav3.3 channels. To address this question, a series of chimeric channels between Cav3.1 and Cav3.3 channels were constructed and expressed in Xenopus oocytes. Kinetic analysis showed that the slow activation and inactivation kinetics of the Cav3.3 channel were not completely abolished by substitution with any one portion of the Cav3.1 channel. Likewise, the Cav3.1 channel failed to acquire the slow kinetics by simply adopting one portion of the Cav3.3 channel. These findings suggest that multiple structural elements contribute to the slow kinetics of Cav3.3 channels.

Calcium influx via T-type Ca 2ϩ channels immediately causes depolarization of the plasma membrane and a rise in intracellular calcium. The calcium rise can mediate diverse physiological functions such as hormone secretion, neurotransmitter release, smooth muscle contraction, fertilization, and cell differentiation (1)(2)(3). T-type channels are known to participate in pacemaker activities of heart and many neurons including the thalamic neurons (4 -6). Abnormal expression of T-type channels has been implicated in certain pathophysiological conditions such as cardiac hypertrophy and absence epilepsy (7,8).
T-type channels are distinguished from high voltage-activated (HVA) 1 Ca 2ϩ channels by their unique biophysical properties, including low voltage activation, fast activation and inactivation kinetics that produce a criss-crossing pattern between successive traces of a current-voltage (IV) protocol, slow deactivation kinetics, and tiny single channel conductance (2, 9 -11). Molecular cloning studies have revealed heterogeneity of T-type channels with the cloning of ␣ 1 subunits from three genes (Ca v 3.1, ␣ 1G ; Ca v 3.2, ␣ 1H ; and Ca v 3.3, ␣ 1I ).
Expression studies found that Ca v 3.3 channels generate currents with much slower activation and inactivation kinetics than Ca v 3.1 and Ca v 3.2 channels, which show the more typical transient kinetics described for native T-type channels (2,(12)(13)(14)(15).
The inactivation processes of voltage-dependent ion channels including Na ϩ , K ϩ , and HVA Ca 2ϩ channels play important roles by preventing continual depolarization, hyperpolarization, and overloading of calcium inside cells, respectively. The fast inactivation process of Na ϩ channels is attributed to blocking of the ion conduction pathway by the IFM (Ile-Phe-Met) motif contained in the intracellular loop between domains III and IV (16,17). A similar process occurs in Shaker K ϩ channels, except in this case the inactivation ball is composed by the first 20 amino acids of the N terminus (18,19). In contrast to these channels, voltage-dependent inactivation of HVA Ca 2ϩ channels appears to be affected by multiple regions, including the S-6 regions in domains II, III, and IV (20 -24), the I-II loop (25)(26)(27)(28), and the N (29) and C termini (30 -32). The kinetics of T-type channels resemble those of Na ϩ channels, albeit on a slower time scale, suggesting that they may also inactivate by a ball-and-chain mechanism. However, preliminary evidence indicates that T-type channels inactivate by similar processes as HVA Ca 2ϩ channels. For example, Marksteiner et al. found that both channel families contain important inactivation determinants in the domain III S6 segment (33). Similarly, intracellular loops also play a role in T-type channel inactivation, as evidenced by the alternatively spliced variants of the III-IV loop in Ca v 3.1 and the C terminus in Ca v 3.3 (34 -36). Chimeric studies using Ca v 3.1 and Ca v 1.2 (L-type) channels identified a negatively charged region in the C terminus as critical for fast inactivation of Ca v 3.1 channels (37). The goal of the present study was to identify structural determinants that confer slow kinetics onto Ca v 3.3 channels using chimeras with Ca v 3.1. The main finding of these studies is that the more donated by Ca v 3.3 to the chimeric channels, the more Ca v 3.3-like the currents became. These findings suggest that there is no single inactivation ball, but as for HVA channels, there are multiple structural elements that contribute to inactivation of low voltage activation channels.

Construction of Ca v 3.1/Ca v 3.3 Chimeric Ca 2ϩ Channels
The cloning of the rat Ca v 3.1 (GenBank TM accession number AF027984) and Ca v 3.3 (GenBank TM accession number AF086827) was reported previously (12,14). Chimeric channel cDNAs were constructed from modifications of cDNAs encoding the Ca v 3.1 (␣ 1G ) and Ca v 3.3 (␣ 1I ) channels.
The plasmids carrying chimeric cDNAs are composed of the following fragments. In some cases, restriction enzyme site(s) were introduced by PCR mutagenesis using modified primers and Pfu DNA polymerase. All fragments derived from PCR were verified by sequence analysis. The restriction enzyme sites are marked by numbers in parentheses by indicating 5Ј-terminal nucleotide generated by cleavage. The origins of the fragments are also denoted in the parentheses. Silent and nonsilent restriction sites were generated by PCR and are indicated by asterisks and crosses, respectively. The locations of the borders in the Ca v 3.1 and Ca v 3.3 chimeras are shown below and schematically represented in Fig. 2.

Expression of the Ca v 3.1, Ca v 3.3, and Chimeras in Xenopus Oocytes
The cDNA constructs were linearized at the 3Ј end by NotI or AflII. Transcripts were synthesized in vitro using T7 RNA polymerase according to the supplied protocol (Ambion, Austin, TX). The concentrations of the synthesized cRNAs were measured spectrophotometrically.

Electrophysiological Recordings in Oocytes and Data Analysis
Barium currents were measured at room temperature 4 -8 days after cRNA injection using a two-electrode voltage-clamp amplifier (OC-725C, Warner Instruments, Hamden, CT). Microelectrodes (Warner Instruments) were filled with 3 M KCl, and their resistances were 0.2-1.0 M⍀. The 10 mM Ba 2ϩ bath solution contained 10 mM Ba(OH) 2 , 90 mM NaOH, 1 mM KOH, 5 mM HEPES (pH 7.4 with methanesulfonic acid). The currents were sampled at 5 kHz and low pass-filtered at 1 kHz using the pClamp system (Digidata 1320A and pClamp 8; Axon instruments, Foster City, CA). Peak currents and exponential fits to currents were analyzed using Clampfit software (Axon Instruments), and a graphical presentation of the data was obtained using Prism software (GraphPad, San Diego, CA). The data are presented as the means Ϯ S.E. The data were tested for significance using Student's unpaired t test with p Ͻ 0.05, p Ͻ 0.01, and p Ͻ 0.001 as the levels of significance.

RESULTS
Functional expression of Ca v 3.1 and Ca v 3.3 channels was measured using the Xenopus oocyte expression system. Their biophysical properties were consistent with previous findings, with Ca v 3.1 generating quickly inactivating currents and Ca v 3.3 generating slowly inactivating currents (12,14). Fig. 1A illustrates representative traces of the Ca v 3.1 and Ca v 3.3 currents evoked during a test pulse to Ϫ20 mV. At this potential, The currents were measured in Xenopus oocytes using the two-electrode voltage-clamp method with 10 mM Ba 2ϩ as charge carrier. The currents were elicited by test pulses to varying voltages from a holding potential of Ϫ90 mV. A, typical Ca v 3.1 and Ca v 3.3 channel currents evoked by a test pulse to Ϫ20 mV. The currents were superimposed after normalizing the current amplitudes to the peak values. B and C, average activation (B) and inactivation (C) time constants of current traces evoked by a test pulse to Ϫ20 mV. When the currents were fit with two exponentials, the activation constants were 1.4 Ϯ 0. the activation and inactivation rates of Ca v 3.1 currents were about 10-fold faster than those of the Ca v 3.3 currents (Fig. 1, B and C). Significant kinetic differences were also observed at other test potentials (Fig. 1, D and E).
To determine the structural element(s) in Ca v 3 channels that lead to these striking kinetic differences, nine chimeras between the Ca v 3.1 and Ca v 3.3 channels were constructed and expressed in Xenopus oocytes. As shown in Fig. 2, all of the chimeras were constructed by substitution of certain regions of the Ca v 3.3 with the corresponding regions of the Ca v 3.1 or vice versa. Then we compared the kinetic properties of the nine chimeras with those of the parent channels. Recently, Staes et al. (37) have reported that substitution of the Ca v 3.1 C terminus with the same region of Ca v 1.2 dramatically slowed down the inactivation kinetic of the chimeric channel currents, suggesting that the C terminus of Ca v 3.1 channels contains a critical element that mediates fast inactivation. Accordingly, we first compared the kinetics of GI ⌬C and IG ⌬C channels constructed by reciprocal exchanges of the C termini of Ca v 3.1 and Ca v 3.3 (Fig. 2). Fig. 3A illustrates representative current traces of these C-terminal chimeras recorded during test pulses to Ϫ20 mV and then normalized for comparison. On average, activation and inactivation time constants of the GI ⌬C currents were 2.0 Ϯ 0.3 and 15.4 Ϯ 0.8 ms, whereas those of the Ca v 3.1 were 1.4 Ϯ 0.1 and 7.2 Ϯ 0.4 ms, respectively. Thus, substitution of the Ca v 3.1 C terminus with that of Ca v 3.3 produced a 2-fold slowing of the inactivation rate, with a negligible effect on the activation rate (Fig. 3, B and C). Conversely, substitution of the Ca v 3.3 C terminus with that of Ca v 3.1 (IG ⌬C ) slowed activation by 72% and accelerated inactivation by 32% (39.3 Ϯ 5.6 and 66.1 Ϯ 0.9 ms, respectively) when compared with Ca v 3.3 currents (22.8 Ϯ 1.5 and 97.0 Ϯ 3.8 ms). The difference in current kinetics was greater at hyperpolarized test potentials, whereas the voltage-independent rates observed at depolarized potentials were more similar (Fig. 3, D  and E).
To assess the contribution of the intracellular loops to current kinetics, three loop chimeras, GI ⌬12L , IG ⌬12L , and IG ⌬23L , were constructed (Fig. 2). Fig. 4A illustrates normalized peak currents of the loop chimeras. Unexpectedly, the activation and inactivation of GI ⌬12L currents became faster than those of Ca v 3.1 (Fig. 4, D and E). Replacement of the I-II loop in Ca v 3.3 (IG ⌬12L ) exerted little effect on inactivation kinetics compared with Ca v 3.3 (Fig. 4C). However, activation of IG ⌬12L was much faster than Ca v 3.3 and IG ⌬23L at most voltages tested (Fig. 4D). In the case of IG ⌬23L , inactivation kinetics were significantly slower than Ca v 3.3 and IG ⌬12L channels, whereas activation kinetics were slightly faster than Ca v 3.3 and much slower than IG ⌬12L channels (Fig. 4, C and E). Overall, switching of any loop failed to produce a dramatic transition from a slow phenotype to a fast one and vice versa. Thus, it is unlikely that either the I-II or the II-III loop alone is a major structural element determining the activation and inactivation kinetics of T-type channels.
Next, we tested whether kinetics are controlled by multiple structural regions including both membrane spanning domain(s) and intracellular loop(s). Chimeric channels containing each half of Ca v 3.1 and Ca v 3.3, denoted as IG ⌬12D and IG ⌬34D (Fig. 2), were constructed and expressed in Xenopus oocytes. As illustrated in Fig. 5, both chimeras displayed similar inactivation kinetics with time constants that lie halfway between those of the Ca v 3.1 and Ca v 3.3 currents. These results suggest that no single half portion determines inactivation kinetics and that each half contributes equally. Notably, IG ⌬34D displayed much faster activation kinetics than IG ⌬12D at low voltage ranges. This finding suggests that the III-IV domain might play a more critical role than the I-II domain of Ca v 3.1 in determining activation kinetics.
Because the experiments using the half:half chimeric channels failed to localize a specific gating particle that determines activation and inactivation kinetics, we expanded the study to three-quarter:quarter chimeras (IG ⌬234D , IG ⌬34D , and IG ⌬4D ). Each chimera derived from Ca v 3.3 contained different structural regions of the Ca v 3.1 channel, i.e. domain IV (IG ⌬4D ), domains III and IV (IG ⌬34D ), or domains II through IV (IG ⌬234D ; Fig. 2). As illustrated in Fig. 6, increasing the proportion of Ca v 3.1 in the chimera steadily shifted the kinetic phenotype toward Ca v 3.1. These results also show that multiple structural elements are involved in determining the kinetics of Ca v 3 channels.
Normalized current-voltage relationships of the Ca v 3.1, Ca v 3.3, and different chimeric channels were compared in Fig. 7A. Although the thresholds for all of the chimeras were around Ϫ60 mV, differences were noted in the positions of their peak currents. Maximum current amplitudes were detected at potentials ranging between Ϫ28 and Ϫ16 mV in the following order: The thresholds and peak currents of most chimeras tend to be slightly shifted toward negative potentials when compared with their parent channels (Ca v 3.1 and Ca v 3.3), whereas those of IG ⌬12D and IG ⌬C are shifted toward positive potentials. These results were quantitated by calculating the chord conductance and fitting these data with a Boltzmann equation (Fig. 7B). Similar shifts were observed in the steady-state inactivation curves of these channels (Fig. 7C). The mid-points of steady-state inactivation and activation are presented in Table I. DISCUSSION Although all three recombinant Ca v 3 channels form robust low voltage activation channels, the Ca v 3.3 (␣ 1I ) currents display slow kinetics that markedly differ from the other two isoforms, Ca v 3.1 (␣ 1G ) and Ca v 3.2 (␣ 1H ) (14,15). The goal of our study was to determine the structural elements(s) responsible for these differences. To address this question, various G-I chimeric channels were constructed and heterologously expressed in Xenopus oocytes. The major finding in this study was that structural elements conferring the slow kinetics of Ca v 3.3 currents or the fast kinetics of Ca v 3.1 currents are not restricted but spread over the whole ␣ 1 subunit.
Reciprocal switching of the C terminus between Ca v 3.1 and Ca v 3.3 channels partially conferred the original inactivation properties of the parent channels to the chimeras (Fig. 3). Previously, Staes et al. (37) proposed that the C terminus contains a negatively charged region that is critical for determining the fast inactivation kinetics of Ca v 3.1 currents. However, sequence alignment reveals that the negatively charged residues are well conserved in the same positions of Ca v 3.1 and Ca v 3.3. Thus, it remained a possibility that the nonconserved portion of the C terminus might determine subtype-specific kinetics. This hypothesis is supported by the recent finding that splice variation at exon 33 in the C terminus of Ca v 3.3 channels alters kinetics (35,36). In contrast, there were few kinetic effects found in rat splice variants of exon 34 that delete most of the C terminus (36). The fact that substitution of the C terminus induced just partial changes in current kinetics led us search for additional structural elements.
Our guiding hypothesis was that introduction of different regions from a slow channel into a fast channel might transfer the slow phenotype. Therefore it was quite surprising that the introduction of the I-II loop from Ca v 3.3 into Ca v 3.1 produced the opposite result and accelerated current kinetics (Fig. 4). Similarly, introduction of the Ca v 3.1 I-II loop into Ca v 3.3 conferred faster activation kinetics (IG ⌬12L ; Fig. 4). Although these results cannot be explained simply, they do indicate that the I-II loop does not contain the structural region that confers subtype-specific kinetics and suggest other structural portion(s) are also required for establishing channel kinetics. This notion is supported by the fact that substitution of the Ca v 3.1 II-III loop with the corresponding region of Ca v 3.3 decreased the activation time constants but had the opposite effect on inactivation time constants (Fig. 4). In the case of HVA Ca 2ϩ channels, auxiliary ␤ subunits (25,(27)(28), G-proteins (26,38), and syntaxin (39,40) were found to potently modulate the gating of currents by interacting with intracellular loops of the channels. However, conserved binding sites of ␤ subunits, G-proteins, or syntaxin were not found in the intracellular loops of Ca v 3 channels. Nevertheless, it is still possible that unidentified auxiliary subunits and diverse signaling molecules including enzymes can regulate T-type channel kinetics. These possible mechanisms might be supported by the following two examples. It is known that the activation and inactivation kinetics of Ca v 3.3 channels are quite different depending on the heterologous expression systems used (14,36). Recently, we also observed that the current kinetics of Ca v 3.3 channels expressed in HEK cells could be modulated after activation of M1 muscarinic receptors. 2 In voltage-activated Na ϩ channels, the IFM motif of the III-IV loop has been found to play a pivotal role as an inactivation ball (16,17). However, IFM-like sequences are not present in the III-IV loops of Ca v 3 channels. The III-IV loops of all three Ca v 3 channels share high sequence similarity, and their lengths are also similar. According to recent reports (2, 34), three Ca v 3.1 splice variants in the III-IV loop were found, and their expressed currents were not profoundly different in terms of their biophysical properties. Moreover, replacement of the III-IV loop of Ca v 3.1 with the corresponding portion of a slowly inactivating Ca v 1.2 had little effect on inactivation kinetics (37). These findings indicate that the III-IV loops of Ca v 3 channels do not contain an inactivation ball as described for Na ϩ channels.
Our results show that replacement of any of the cytoplasmic loops could not confer kinetics from the parental to the donor channel. This finding led us to examine domain(s) including membrane-spanning portions as well as loops, using half-half chimeras (IG ⌬12D and IG ⌬34D ) and serial chimeras (IG ⌬234D , IG ⌬34D , and IG ⌬4D ). A general pattern obtained from comparing the current kinetics of the two parent and chimeric channels is that the more Ca v 3.3 donated to the chimeric channels, the more Ca v 3.3-like its kinetics became. This pattern can be more easily appreciated by plotting the percentage of amino acids from Ca v 3.3 in the chimeric channels versus the inactivation or activation time constants (Fig.  8). The general pattern strongly supports our main conclusion that structural elements that determine the kinetics of Ca v 3 channels are not localized but distributed over the whole structure.

3, and chimeras
Relative currents were fit with the Boltzmann equation to determine the midpoint of voltage dependence (V 50 ) and slope (k). Time constants of activation ( act ) and inactivation ( in ) were obtained from double exponential fits to the current traces measured during test pulse to Ϫ20 mV. The data are shown with the means Ϯ S.E.M, and the number of oocytes tested appears in parentheses.