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Am J Physiol Cell Physiol 292: C1078-C1086, 2007. First published October 4, 2006; doi:10.1152/ajpcell.00353.2006
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NERVOUS SYSTEM CELL BIOLOGY

Altered frequency-dependent inactivation and steady-state inactivation of polyglutamine-expanded {alpha}1A in SCA6

Haiyan Chen and Erika S. Piedras-Rentería

Department of Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois

Submitted 26 June 2006 ; accepted in final form 2 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease of the cerebellum and inferior olives characterized by a late-onset cerebellar ataxia and selective loss of Purkinje neurons (15, 16). SCA6 arises from an expansion of the polyglutamine tract located in exon 47 of the {alpha}1A (P/Q-type calcium channel) gene from a nonpathogenic size of 4 to 18 glutamines (CAG4–18) to CAG19–33 in SCA6. The molecular basis of SCA6 is poorly understood. To date, the biophysical properties studied in heterologous systems support both a gain and a loss of channel function in SCA6. We studied the behavior of the human {alpha}1A isoform, previously found to elicit a gain of function in disease (41), focusing on properties in which the COOH terminus of the channel is critical for function: we analyzed the current properties in the presence of beta4- and beta2a-subunits (both known to interact with the {alpha}1A COOH terminus), current kinetics of activation and inactivation, calcium-dependent inactivation and facilitation, voltage-dependent inactivation, frequency dependence, and steady-state activation and inactivation properties. We found that SCA6 channels have decreased activity-dependent inactivation and a depolarizing shift (+6 mV) in steady-state inactivation properties consistent with a gain of function.

trinucleotide repeats; ataxia; calmodulin

VOLTAGE-GATED P/Q-type calcium (Cav2.1) channels play an important role in mediating neurotransmitter release in the nervous system, postsynaptic integration, neuroplasticity, neural excitability, and gene transcription (30, 34, 40, 53). P-type currents were first described in cerebellar Purkinje neurons and are characterized by their slow inactivation and their high sensitivity to the funnel web spider venom {omega}-agatoxin-IVA with an IC50 < 1 nM (35). Q-type currents, on the other hand, were identified in cerebellar granule neurons, have fast inactivation kinetics, and are less sensitive to {omega}-agatoxin IVA (IC50 = ~100–200 nM) (44, 48, 52). Currents with intermediate properties between P and Q have been described in several systems (12, 33, 44, 56, 61). All these currents are generated by channels containing the principal subunit {alpha}1A, encoded by the CACNA1A gene; the combination of different {alpha}1A splicing variants and/or diverse auxiliary beta subunits can contribute to the different current phenotypes observed in neurons (2, 28, 51).

Splicing mechanisms can give rise to several {alpha}1A isoforms, with seven identified loci in humans (27, 51). Splicing in the last locus in exon 47 can originate two different {alpha}1A isoforms, one with a short COOH-terminal tail ({Delta}47) or a channel with a longer (250 amino acids) COOH-terminus (+47) (22, 38, 51, 63); the latter isoform represents ~65% of the message in normal human cerebellum (51).

The neurodegenerative disorder spinocerebellar ataxia type 6 (SCA6) occurs by the expansion of a polyglutamine (polyQ) tract located in exon 47 (63) from a normal trinucleotide tract size of 4–18 (CAG4–18) to 19–33 repeats in the disease (13, 64). SCA6 is characterized by late-onset cerebellar ataxia and particularly Purkinje neuron death (15, 16). However, the molecular basis of SCA6 is poorly understood. Heterologous studies have shown conflicting results, either a gain of function (41, 45) or a loss of function (32, 54) of the SCA6 mutant proteins. The divergent results obtained for steady-state activation (SSA) and steady-state inactivation (SSI) parameters are shown in Table 1; Restituito et al. (45) also reported that SCA6 {alpha}1A (CAG30) showed a slower kinetics of inactivation compared with wild type (WT). The discrepancies among the studies are thought to be caused by the use of different experimental systems, different auxiliary subunits, and/or different {alpha}1A splicing isoforms and sources.


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  		Table 1. Reported biophysical properties of WT and SCA6 {alpha}1A

 
In the present study we focused on the analysis of channel properties that involve the COOH terminus of {alpha}1A, a segment in which many molecules can interact with and modulate the channel function; we tested whether properties mediated by calmodulin (CaM) and auxiliary beta-subunits were disrupted in the polyQ-expanded SCA6 {alpha}1A. We assayed the current inactivation kinetics (R800) and also the voltage-dependence inactivation properties of WT and SCA6-expanded {alpha}1A expressed in HEK-293 cells, focusing on the effects of the auxiliary beta2a- and beta4-subunits. Both subunits have a secondary binding site at the COOH terminus of {alpha}1A in addition to the universal binding site of beta-subunits located at the I–II loop of {alpha}1A (6, 42). Functionally, the coexpression of {alpha}1A with beta4 generates currents with Q-like kinetics, whereas beta2a can confer a P-like kinetic phenotype (8, 46); both subunits are expressed in the cerebellum, with the beta4-subunit being the most abundant in this tissue (31).

We also studied calcium-dependent facilitation (CDF) and inactivation (CDI) properties of all constructs as markers for {alpha}1A modulation by CaM. CDF is an important modulation mechanism for calcium channels, thought to play a role in neuronal burst firing and other frequency-dependent activities, and CaM is a major modulator of these phenomena (5, 7, 20, 25, 66). CaM is constitutively bound to calcium channels and functions practically as another subunit of the channel complex, modulating its function (11). CaM preassociates to the COOH terminus of {alpha}1A and mediates calcium-dependent facilitation via the activation of its COOH-terminal lobe upon local calcium entry. Subsequent global calcium increases lead to the activation of the NH2-terminal lobe of CaM, resulting in calcium-dependent inactivation (5, 26).

Current activation kinetics ({tau}on), frequency-dependent and steady-state properties of WT and SCA6 mutant channels also were analyzed. We found SCA6 channels have current activation, inactivation kinetics, and calcium-dependent facilitation properties similar to those of WT {alpha}1A; however, clear differences in frequency-dependent and SSI properties were consistently found in SCA6 {alpha}1A. Decreased frequency-dependent inactivation and a depolarizing shift in SSI were detected for the SCA6 isoforms tested, CAG23 and CAG72, compared with CAG11. Our data support a gain of function of {alpha}1A in SCA6.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
HEK-293 Cell Culture

HEK cells were obtained from American Type Culture Collection (Manassas, VA) and kept in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1,000 U/ml penicillin-streptomycin. Cells were kept in a 5% CO2-humidified atmosphere at 37°C and split every 2–3 days.

DNA Preparation and Transfection

Human {alpha}1A cDNA was obtained as previously described (41). Control (WT) channels with normal polyQ tract size (+47, CAG11) were compared with the expanded {alpha}1A SCA6 channel [+47, CAG23 (23 glutamine repeats)] and/or an exaggerated expansion (+47, CAG72). CAG72 is not found clinically, but it was used to maximize the detection of possible subtle changes that might go undetected in the naturally expanded channels.

We studied the most abundant {alpha}1A splice variant in human cerebellum, isolated from SCA6 patients and control individuals (63), encoding for the variants with exons [{Delta}10A, +16/17, {Delta}17A, –31 (–NP), +37a, +43/+44, +47]. {alpha}2{delta}- and beta-subunits obtained from rabbit and rat, respectively, were kindly supplied by Dick Tsien's and David Yue's laboratories.

Channels were transfected using the calcium phosphate method (39). HEK-293 cells were grown to 60–80% confluency in 60 x 15-mm culture dishes and plated into 12-mm-diameter round coverslips; only HEK cells from the 10th–50th passages were used. The cells were transfected with 3 µg of +47 {alpha}1A, with auxiliary {alpha}2{delta}- and beta4- or beta2a- subunits 24 h after being split. The stoichiometry of {alpha}1A:{alpha}2{delta}:beta4/beta2a was 1:1.3:1.3. The DNA amounts were carefully quantified before transfection by UV spectroscopy and gel electrophoresis.

Electrophysiology

Currents were recorded at room temperature by using the whole cell patch-clamp configuration with an Axopatch 200B patch-clamp amplifier (Axon). pCLAMP 8.2 was used to acquire and analyze data. Signals are filtered at 2 kHz and digitized at 10 kHz. Recording pipettes had resistances of 3–6 M{Omega}, and series resistance was <15 M{Omega}, 80% compensated. Currents were measured in 5 mM extracellular Ca2+ (5 Ca) or Ba2+ (5 Ba) as the charge carrier containing (in mM) 5 CaCl2 or 5 BaCl2, 140 TEA-Cl, 10 HEPES, and 10 glucose (pH 7.4, 300 mosmol/l). The intracellular solution contained (in mM) 108 CsMeSO3, 4 MgCl2, 9 HEPES, 15 creatine phosphate-Tris, 1 or 0.5 EGTA-Cs, 1 GTP-Li, and 5 ATP-Mg (pH 7.4, 265 mosmol/l). Free calcium concentration was measured using Maxchelator software, downloaded from Dr. Chris Patton's website (http://www.stanford.edu/~cpatton/maxc.html). Cell capacitance was measured from the transient current elicited by a 5-mV depolarizing voltage pulse from a holding potential (Vh = –90 mV). The junction potential between the 5 Ca and 5 Ba recording solution was 1.9 mV (not corrected).

Current activation kinetics. Time constants of activation ({tau}on) were measured by fitting the activation phase of the currents with the best exponential fit. All the currents were fit with single exponential except for two currents expressing beta2a (see GoFig. 2), which were best fit with a double exponential. In these two cases, the fast-activating component was used, because it was very similar to the values seen in single-exponentially fitted cells.


Figure 1
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  		Fig. 1. Calcium- and voltage-dependent inactivation properties are indistinguishable between wild type (WT) and spinocerebellar ataxia type 6 (SCA6) {alpha}1A-subunits, irrespective of the beta-subunit coexpressed or the intracellular calcium buffering level. A and B: examples of representative Q-like (A) and P-like currents (B) generated from the cells cotransfected with trinucleotide CAG11 (solid trace) or CAG23 (shaded trace) {alpha}2{delta} and beta4 (A) or beta2a (B). Currents were evoked with a 1-s-long test pulse from –90 to 0 mV. C: current inactivation kinetics (R800 values) for CAG11 and CAG23 coexpressed with beta4-subunits measured in the presence of 1 mM EGTA (top row). Recordings made in 5 mM extracellular Ca2+ (5 Ca) are shown at left and in 5 mM Ba2+ (5 Ba) at right. Middle row, R800 values measured in the presence of beta2a and 1 mM EGTA; bottom row, R800 values measured in the presence of beta4 and 0.5 mM EGTA (P > 0.16; n = 6–14 for all experiments).

 

Figure 2
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  		Fig. 2. Time constant of activation ({tau}on) is similar in WT and SCA6 {alpha}1A. A: normalized current-voltage (I-V) relationship of CAG11, CAG23, and CAG72. Currents were recorded in 5 Ca. B: example of representative currents (solid traces); the single exponential fit is the shaded trace. C: {tau}on values for CAG11 and CAG23 coexpressed with beta4-subunits recorded in the presence of 1 mM EGTA (top row). Traces recorded in 5 Ca are shown at left (1.49 ± 0.16 for CAG11 and 1.31 ± 0.12 for CAG23 at 10 mV) and in 5 Ba at right (1.45 ± 0.08 and 1.32 ± 0.09 at 0 mV, respectively) (P > 0.05, n = 5 to 18). Middle row, {tau}on values recorded with beta2a + 1 mM EGTA in 5 Ca (1.30 ± 0.11 and 1.16 ± 0.11, respectively) and 5 Ba (1.31 ± 0.05 and 1.20 ± 0.15, respectively); bottom row, {tau}on values for CAG11, CAG23, and CAG72 recorded with beta4 + 0.5 mM EGTA in 5 Ca (1.04 ± 0.07, 1.05 ± 0.06, and 1.03 ± 0.04, respectively) and 5 Ba (1.24 ± 0.11, 1.22 ± 0.12, and 1.21 ± 0.06, respectively) (P > 0.17, n = 5–26 for all experiments).

 
Current inactivation kinetics. The currents were elicited by stepping membrane potential to various test voltages (Vtest = –60 to +50 mV, {Delta}V = 10 mV) from Vh = –90 mV for 1 s. The relative inactivation index (R800) was calculated as the ratio of the current amplitude measured at 800 ms divided by the peak current for each voltage.

Steady-state activation. SSA was determined by stepping the membrane potential to various prepulse voltage levels (Vpp = –60 to +60 mV, {Delta}V = 10 mV) for 150 ms from Vh = –90 mV before repolarizing to a fixed Vtest = –60 mV to evoke the tail currents. Tail current peaks were normalized to the maximal value.

Steady-state inactivation. SSI was determined by stepping the membrane potential to various prepulse voltage levels (Vpp = –100 to +40 mV, {Delta}V = 10 mV) for 2 s before depolarization to a fixed Vtest = +10 mV to evoke channel opening (55). Peak currents obtained at all voltages were normalized to the maximal value. Both the steady-state activation and inactivation curves were fitted with a single Boltzmann function of the form Imax/[1 + exp{(V1/2 – V)/k}] + m, where Imax is the maximal current, V1/2 is the half-maximal voltage of activation or inactivation, k is the slope factor, and m is the pedeatal factor.

Calcium-dependent facilitation. CDF was measured using standard square pulse protocols as described by Dr. D. T. Yue's laboratory (7). Nonfacilitated currents generated in the absence of a prepulse (–pp) elicit an initial fast component followed by a slow phase of calcium current increase. The initial, fast activation reflects the normal gating mode of the channel, followed by a calcium-induced conversion to a facilitated gating mode with enhanced open probability. When a short square pulse (20 ms) that elicits negligible inactivation is given as a prepulse (+pp), the channels are prefacilitated so that a subsequent pulse activates currents more rapidly, from an already facilitated state. The relative facilitation (RF = {Delta}Q/{tau}), or the fraction of channels prefacilitated by the prepulse (Ffacilitated), was measured as the integral of the difference in charge ({Delta}Q, see shaded area in Fig. 3B) carried out in the presence and absence of the prepulse, divided by the time constant of facilitation. The time constant of facilitation {tau} was determined from the traces with +pp. The RF calculation assumes that all the channels are originally in a normal mode at the onset of the test pulse and that the shift to facilitated mode occurs monoexponentially with a time constant {tau}; then RF = Ffacilitated x Po,normal/Po,facilitated, where Po,normal and Po,facilitated are the steady-state open probabilities at normal and facilitated modes (7). This experiment was repeated in the presence of 5 Ba to determine the contribution of the calcium-independent, voltage-dependent facilitation component, which was then subtracted from the value obtained in the presence of extracellular calcium, where both voltage- and calcium-dependent components are present to obtain the calcium-dependent component (g = RFCa – RFBa).


Figure 3
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  		Fig. 3. Calcium-dependent facilitation (CDF) is similar between WT and SCA6 {alpha}1A. A: pulse protocol used to measure CDF. Currents elicited with and without a prepulse (pp) to 0 mV were normalized to the values at the end of the pulse. B: example of representative currents. Shaded area represents the difference in charge ({Delta}Q; see MATERIALS AND METHODS). C: average relative facilitation (RF = {Delta}Q/{tau}) in 5 Ca (solid symbols) or 5 Ba (open symbols) for CAG11 (circles), CAG23 (triangles), or CAG72 (squares) plotted against the different prepulse voltages. CDF index (g = RFCa – RFBa) was measured in the presence of beta4 + 1 mM EGTA (1.8 ± 0.2 and 2.4 ± 0.4 for CAG11 and CAG23, first row), beta2a + 1 mM EGTA (1.3 ± 0.3 and 1.7 ± 0.3, second row), or beta4 + 0.5 mM EGTA (1.1 ± 0.5, 1.4 ± 0.5, and 2.0 ± 0.5 for CAG11, CAG23, and CAG72, third and fourth rows) (P > 0.10, n = 6–10 for all experiments).

 
Action potential waveforms. Frequency-dependent inactivation (FDI) and frequency-dependent facilitation (FDF) were studied using the more physiological action potential waveforms (APW) compared with the classic square pulse protocols. APW were constructed from a neuronal action potential and digitized at frequencies of 10 and 100 Hz. The inactivation ratio (IR) was measured by dividing the amplitude of the last spike by the initial response at each frequency (APWend/APW1; where 1 and end are the first and last measured spikes). The facilitation ratio (FR) is described as the amplitude of the peak response (APWmax) divided by the peak value of the first spike (APW1).

Data Analysis

Student's t-test (one tail, homoscedastic) was applied for statistical analysis in this study. Values are represented as means ± SE; P < 0.05 is considered as indicating statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
R800 is Similar Between WT and SCA6 Channels

The inactivation kinetics of currents generated with either WT (CAG11) or SCA6 {alpha}1A (CAG23 and/or CAG72) were first tested when coexpressed with the auxiliary beta4-subunit, which generated fast-inactivating Q-like currents, as expected (Fig. 1A). The index of current inactivation, R800 (shown in Fig. 1C) did not change between CAG11 and CAG23 in the presence of 5 Ca (left) [R800 = 0.31 ± 0.03 for CAG11 (n = 12) and 0.30 ± 0.02 for CAG23 (n = 12) measured at 10 mV (P > 0.17)]. Similarly, no changes were observed when barium (5 Ba) was used as the charge carrier (right) [R800 = 0.33 ± 0.04 for CAG11 and 0.35 ± 0.02 for CAG23 (n = 9 and 14, respectively) measured at 0 mV (P > 0.33)]. In general, currents measured in the presence of calcium reflect the degree of inactivation induced by both calcium and voltage, whereas currents recorded in the presence of barium monitor the voltage-dependent inactivation properties only (20). The experiments were done in the presence of 1 mM EGTA intracellular solution, which yield a free calcium concentration ([Ca2+]free) of 6.4 nM. To avoid the possibility that potential variations in kinetics between WT and SCA6 {alpha}1A were undetected because of the fast-inactivating nature of currents coexpressed with beta4-subunit or the slightly supraphysiological calcium-buffering levels used, we repeated these measurements in the presence of 1) beta2a-subunits, which yield characteristic slow-inactivating currents reminiscent of native P-type currents (Fig. 1C, middle row) and 2) beta4 + 0.5 mM EGTA, a concentration of intracellular EGTA that is reported to be within the physiological calcium buffer concentration range (26) (Fig. 1C, bottom row). None of these conditions uncovered differences between WT and SCA6. R800 values obtained with beta2a were 0.51 ± 0.04 for CAG11 (n = 8) and 0.53 ± 0.04 for CAG23 (n = 7) in 5 Ca (left; P > 0.34) and 0.72 ± 0.03 for CAG11 (n = 9) and 0.67 ± 0.05 for CAG23 (n = 6) in 5 Ba (right; P > 0.16). Data obtained using beta4 + 0.5 mM EGTA also included data for CAG72. Again, R800 values were similar for the three constructs: 0.15 ± 0.02 for CAG11 (n = 16), 0.15 ± 0.02 for CAG23 (n = 11), and 0.15 ± 0.03 for CAG72 (n = 10) for 5 Ca (left; P > 0.34), and 0.21 ± 0.03 for CAG11 (n = 11), 0.23 ± 0.03 for CAG23 (n = 12), and 0.23 ± 0.04 for CAG72 (n = 10) for 5 Ba (right; P > 0.35). These results show that the interactions of {alpha}1A with beta4 and beta2a are normal in SCA6 mutants and suggest that the known secondary interaction sites for beta4 and beta2a located at the COOH terminus of {alpha}1A are not hindered by the presence of the expanded polyQ tract.

{tau}on is Similar in WT and SCA6 {alpha}1A

Using the experimental conditions described for Fig. 1, we also tested the current activation kinetics as measured by {tau}on for all the constructs. Current kinetics were measured in either 5 Ca (at 10 mV) or 5 Ba (at 0 mV) when {alpha}1A was expressed with beta4- or beta2a-subunits (values are given in Fig. 2 legend). Currents were evoked with step pulses from Vh = –90 mV to Vtest = –40 to +50 mV with 10-mV increments. In all cases, currents were first observable at –20 mV and reached their peak at 10 mV, as shown in the normalized current-voltage (I-V) relationships in Fig. 2A. {tau}on was measured by best exponential fitting of the activation phase of the currents (shaded line in Fig. 2B) (also see MATERIALS AND METHODS). As shown in Fig. 2C, no differences in {tau}on were detected between CAG11 and CAG23 in all conditions or with CAG72 measured in the presence of beta4 + 0.5 mM EGTA. The change of intracellular calcium buffer EGTA from 1 to 0.5 mM did not affect this outcome either (n = 5–26) (P > 0.17).

CDF Properties of WT and SCA6 {alpha}1A are Indistinguishable

We used a paired-pulse protocol to study CDF (Fig. 3A) with very short square prepulses that elicit negligible inactivation, based on published standard protocols (see MATERIALS AND METHODS) (5, 7). Facilitation values (RF) obtained in 5 Ba and 5 Ca are shown. As shown in Fig. 3C, the g values, or the calcium-dependent facilitation component (RFCa – RFBa) were not statistically different among constructs or when tested in the presence of beta4 + 1 mM EGTA (first row: n = 8 for CAG11, n = 10 for CAG23; P > 0.10), beta2a + 1 mM EGTA (second row: n = 7 for CAG11, n = 7 for CAG23; P > 0.17), or beta4 + 0.5 mM EGTA (third and fourth rows: n = 7, 6, and 8 for CAG11, CAG23, and CAG72, respectively; P > 0.10), although a trend of increased g is seen for CAG23 and CAG72 compared with CAG11 in all conditions (g values are given in Fig. 3 legend).

SCA6 {alpha}1A has Decreased FDI and Increased FDF

We also probed the frequency-dependent properties of SCA6 {alpha}1A by using APW protocols at two low (10 Hz, APW10) and high frequencies (100 Hz, APW100). For this set of experiments, we only studied the combination of beta4-subunit with 0.5 mM EGTA in 5 Ca.

A set of typical currents elicited using low frequencies of stimulation are shown in Fig. 4A, with the stimulation APW protocol depicted above the example currents (CAG72). The parameters used to analyze this experiment are the FR and IR as described in MATERIALS AND METHODS. Facilitation was not observable at this frequency; Fig. 4B shows the inactivation data summary, where a trend of increased IR can be seen in SCA6 compared with WT {alpha}1A. However, these differences did not reach statistical significance [IR = 0.94 ± 0.01 (n = 19) for CAG11 compared with 0.96 ± 0.01 for CAG23 (n = 13) (P > 0.06) and 0.96 ± 0.01 for CAG72 (n = 15) (P > 0.1)].


Figure 4
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  		Fig. 4. Low-frequency-dependent inactivation properties. A: a single neuronal action potential was digitized and delivered at 10 Hz as the stimulus protocol (top); typical responses in 5 Ca are shown at bottom (CAG72). B: inactivation ratio for CAG11, CAG23, and CAG72. APW1 and APW10, peak values of action potential waveform at the first and tenth spikes. No differences were detected between WT and SCA6 {alpha}1A (P > 0.06, n = 19, 13, and 15, respectively).

 
On the other hand, we uncovered remarkable differences between the mutant SCA6 and WT currents when high frequencies of stimulation were used. Figure 5A, top, shows the stimulation protocol used, and a typical response recorded from HEK cells expressing CAG72 {alpha}1A is shown at bottom. Figure 5B shows the response to a single APW. As shown in Fig. 5C, we found substantial changes in the degree of FDI. WT {alpha}1A elicited ~54% of inactivation at the end of the train (IR = 0.46) (n = 19), statistically higher than the values obtained with SCA6 {alpha}1A of 43% for CAG23 (IR = 0.57 ± 0.05, n = 13) (P < 0.02) and 46% in CAG72 (IR = 0.544 ± 0.03, n = 15) (P < 0.03). In addition, as shown in Fig. 5D, WT {alpha}1A elicited a subtle FR of ~2%, as measured by the ratio APWmax/APW1 (1.02 ± 0.01, n = 19). Both CAG23 and CAG72 currents yielded a statistically significant, albeit discrete, increase in facilitation compared with WT (CAG23: 1.04 ± 0.01, n = 13; CAG72: 1.04 ± 0.01, n = 15) (P < 0.04 for CAG23 and P < 0.03 for CAG72).


Figure 5
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  		Fig. 5. SCA6 {alpha}1A elicit less high-frequency-dependent inactivation and increased facilitation than WT. A: 100-Hz APW stimulus protocol (top); typical responses of CAG72 to this stimulus in 5 Ca (bottom). APWmax and APWend, peak values of maximal and last spikes. B: representative responses to a single APW. C: inactivation ratios for CAG11, CAG23, and CAG72 (n = 19, 13, and 15). D: facilitation ratios for CAG11, CAG23, and CAG72 (n = 19, 13, and 15). *P < 0.04 for CAG23 vs. CAG11; *P < 0.03 for CAG72 vs. CAG11.

 
SSI Properties are Altered in SCA6 {alpha}1A

The differences of use-dependent properties between WT and SCA6 described above could arise from alterations in SSI properties (62). In this experiment, we explored the steady-state properties of the channels. First, SSA was assayed with tail currents elicited from Vtest = –60 mV following a 150-ms-long prepulse to various voltages (Vpp = –60 to +60 mV, {Delta}V =10 mV) from Vh = –90 mV. The resulting data were fitted to a single Boltzmann function of the form Imax/[1 + exp{(V1/2 – V)/k}] + m (Fig. 6A; see also Table 2); none of the SSA parameters diverge between WT and SCA6 {alpha}1A. For instance, V1/2 values were ~7 mV for all constructs (P > 0.28); fitted curves are superimposed as continuous lines, whereas the raw data are shown as symbols.


Figure 6
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  		Fig. 6. Steady-state inactivation (SSI) midpoint depolarizing shift in SCA6 {alpha}1A. A: steady-sate activation properties. Results were fitted with a single Boltzmann function, shown as continuous lines superimposed on the empirical values obtained for CAG11 (open circles), CAG23 (shaded triangles), and CAG72 (solid squares). B: SSI properties. C: example currents obtained at +10 mV after an inactivating prepulse at –30 mV (dotted lines in B), normalized to CAG11 currents elicited after a –60-mV prepulse. D: peak responses obtained at 100 Hz (from Fig. 5) plotted against spike number for CAG11 (open circles, n = 19), CAG23 (shaded triangles, n = 13), and CAG72 (solid squares, n = 15).

 

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  		Table 2. Steady-state properties of WT and SCA6 {alpha}1A

 
SSI properties were determined by stepping to a 2-s-long inactivating prepulse to various voltages (Vpp = –100 to +40 mV, {Delta}V = 10 mV) before depolarization to Vtest = +10 mV (55). Normalized currents fitted with a single Boltzmann function are depicted in Fig. 6B. Indeed, the V1/2 values were statistically different between WT and SCA6 {alpha}1A. V1/2 for SCA6 was –27.0 ± 0.4 mV for CAG23 (n = 7) and –28.5 ± 0.2 mV for CAG72 (n = 9), significantly more positive than the value found for the CAG11 (–33.2 ± 0.4 mV, n = 8) (P < 0.006). The slope factor k was –7.6 ± 0.3 fold/mV for CAG23, significantly shallower than for CAG11 (k = –6.5 ± 0.3) (P < 0.05). However, there was no significant difference in k between CAG11 and CAG72 (–6.8 ± 0.2) (P > 0.19) (see Table 2). This result suggests that 1 fold/mV difference in slope seen for CAG23 may not be physiologically relevant.

Figure 6C depicts examples of CAG11 traces elicited by stepping to +10 mV following a prepulse from –30 mV, normalized to the maximal current generated from Vpp = –60 mV (dotted lines in B). As observed from this example, the degree of inactivation in WT (CAG11) channel corresponds to ~56% of the maximal current. In contrast, SCA6 elicited only ~35–40% inactivation compared with control. Similarly, when the FDI data obtained at 100 Hz (from Fig. 5) were plotted against the spike number, the remaining currents at the end of the stimulus were ~43% of the initial spike for WT compared with ~50–52% in SCA6 (Fig. 6D), as predicted by the positive shift in SSI seen in SCA6 {alpha}1A, which may in part underlie this use-dependent phenomenon.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Current Kinetics in SCA6

Both the kinetics of activation ({tau}on) and inactivation (R800) were similar in SCA6 constructs compared with WT {alpha}1A (Figs. 1 and 2). Inactivation strongly depends on modulation by beta-subunits; thus our data show that the secondary binding sites for beta4- and beta2a-subunits located at the COOH-terminal region of {alpha}1A (60) are not affected by the presence of the polyQ expansion in SCA6 {alpha}1A. Moreover, our data do not replicate the previous report that SCA6 {alpha}1A (CAG30) had slower kinetics of inactivation compared with WT (45). The {alpha}1A cDNA isoforms used in both studies were comparable; thus the likely source of discrepancy is the experimental system used (Xenopus oocytes in the former). This highlights the importance of the experimental system and shows that great differences in cellular background exist between the amphibian oocyte system and HEK-293 cells, which are more closely related to neuronal lineages and express several neuron-specific proteins (49).

CDF and CDI as Markers for {alpha}1A Modulation by CaM

CDF is an important modulation mechanism for calcium channels that plays a role in neuronal burst firing and other frequency-dependent activities. We assessed CDI and CDF for SCA6 {alpha}1A for the first time. We used HEK-293 cells, an excellent experimental system that expresses endogenous CaM, which has been used to characterize CaM's interactions with the COOH terminus of {alpha}1A (7). We found that current inactivation kinetics of WT and SCA6 were similar in the presence of either calcium or barium as charge carriers, indicating that neither voltage-dependent nor calcium-dependent components of current inactivation were affected by the presence of the polyQ expansion. Whereas CDF and CDI are modulated by the interactions of {alpha}1A with CaM, auxiliary beta-subunits are a main modulator of voltage-dependent inactivation (VDI), along with portions of intracellular loops (3, 14); our data show that none of these interactions is altered by the presence of the expanded CAG repeats in SCA6. VDI and CDI have been proposed to utilize similar determinants at the EF hand located between the CaM-binding domains in {alpha}1C (20); in accordance with this model, VDI, CDI, and CDF were not affected in SCA6, indicating that access to the COOH-terminal CaM-binding sites and the EF-hand region of {alpha}1A is not hindered by the presence of the expanded polyQ stretch.

Steady-State Properties

SSA properties of WT and SCA6 {alpha}1A did not change in the present study, unlike our previous study (41), in which we detected a significant shift in SSA in CAG23 and CAG72 but not in CAG27. This discrepancy can only be attributed to the beta-subunit used, which is the only experimental difference in the two studies. To approximate physiological conditions, we chose to study beta2a and beta4 in this study (compared with beta1; Ref. 41); both subunits interact with the COOH terminus of {alpha}1A, and beta4 is the most abundant beta isoform and has the highest affinity for {alpha}1A in cerebellum (8, 31), which is the target tissue in SCA6. Clearly, additional experiments in primary cerebellar neurons (where all the isoforms are present) must be done to properly dissect these differences (58).

In the present study we found consistent changes in SSI properties in the two SCA6 mutant channels tested (CAG23 and CAG72), with SSI voltage midpoint values positively shifted by 6 mV in SCA6 consistent with a lower degree of inactivation in SCA6.

FDF and FDI

Neuronal facilitation could also occur via regulation by the neuronal calcium sensor NCS-1 or neuronal calcium-binding protein visinin-like protein-2 (VILIP-2), or by relief of G protein-induced inhibition (1, 7, 24, 57); this and the SSI changes detected in SCA6 led us to test whether FDF and FDI were also affected in SCA6. Facilitation was not detected at low frequencies of stimulation (10 Hz), and no changes in inactivation were observed at this frequency.

High-frequency stimulation (100 Hz) revealed both facilitation and inactivation differences in SCA6. Discrete increases in facilitation were detected in SCA6 (~4% compared with 2% in WT {alpha}1A), and although they were statistically significant, these small changes seem unlikely to be physiologically relevant. The detection of such small levels of high-frequency-dependent facilitation is not surprising given that G protein-mediated inhibition was absent in our cell system and endogenous levels of NCS-1 are negligible in HEK-293 cells (47). Again, it would be interesting to probe these properties against a neuronal background, such as cerebellar granule neurons from {alpha}1A knockout animals, where native {alpha}1A modulators are available (19).

In contrast, more robust differences were detected in the inactivation values; WT {alpha}1A elicited 54% current inactivation at the end of the pulse, 26 and 17% higher than CAG23 (43% inactivation) and CAG72 (46% inactivation), indicating that increased calcium influx occurs in SCA6 under high frequency use. This alteration could be physiologically relevant during cerebellar activity given that Purkinje neurons, the most affected neuron type in SCA6 and the sole output of the cerebellar cortex, have intrinsically high firing rates (17 to 148 Hz) (10, 29, 43). In addition, increases in calcium influx through SCA6 channels could alter neurotransmitter release and downstream cellular events such as neuronal plasticity in general (65).

That the changes in use-dependent inactivation properties could be detected at high but not at low frequencies suggests this phenomenon could be caused by changes in steady-state inactivation properties. Indeed, SSI curves for SCA6 were shifted to depolarizing potentials. For instance, the dotted lines in Fig. 6, B and C, point out inactivation levels seen at –30 mV, which yielded currents ~58% smaller than the normalized CAG11 control and 35–39% smaller in SCA6. These values are comparable to those observed in the frequency dependence experiments (Figs. 5C and 6D), thus indicating that changes in the channel's biophysical property may underlie the use-dependent abnormalities.

Significance

Neurotransmitter release is tightly modulated by calcium entry through neuronal calcium channels, and it is well documented that modulation of P/Q-type can occur via the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complex, such as SNAP25 (59), syntaxin I (50), and synaptotagmin 1 (4, 62), resulting in tighter coupling between calcium influx and the synaptic vesicle machinery. The interaction of P/Q-type channels with a single SNARE protein leads to a hyperpolarizing shift in the SSI of the channel of ~9 mV (62), resulting in reduced availability for opening and inhibition of channel activity, thus reducing random, nonregulated calcium channel openings and vesicle release until channel availability is restored upon association of the entire SNARE complex to the channel. Therefore, altered SSI in SCA6 (+6 mV) could result in abnormal modulation of P/Q-type channel availability in presynaptic sites.

Frequency-dependent changes in SCA6 {alpha}1A also could alter the modulation and timing of neuronal circuits in feedback loops, thus altering the information flow in cerebellar networks and possibly contributing to the ataxic phenotype (36, 37).

Finally, the glutaminopathy component of the disease has been clearly documented. SCA6 mutant protein is detected in cytoplasmic and nuclear aggregates in cerebellar Purkinje neurons from SCA6 patients (17, 18). Furthermore, it was described that COOH-terminal fragments of {alpha}1A 75 kDa in size were toxic in cell line studies and could be detected only when SCA6 {alpha}1A was transfected but not when WT {alpha}1A was present, leading to the suggestion that the polyQ-expanded fragments were more resistant to proteolysis (23). A recent report by Gomez et al. (21) demonstrated that both WT and SCA6 fragments can be detected in cells and that the fragments contain nuclear localization signals that mediate their shuttling into the nucleus, suggesting the possibility that these fragments may act as transcription factors and that the polyQ-expanded version of {alpha}1A may be abnormal in its nuclear function. Whether a channelopathy plays a role in disease is still to be determined; the biophysical differences in SCA6 {alpha}1A determined in the Gomez laboratory and elsewhere tend to be very discrete in nature, consistent with a slow development of disease. Their changes reported were consistently observed in the two SCA6 constructs tested (CAG23 and CAG72), unlike ours and other previous studies, where only one or two of several mutant constructs were different from WT. Certainly, more experiments in cerebellar neuronal systems are needed. We recently reported that apoptotic responses in cerebellar granule neurons are increased in the presence of SCA6 {alpha}1A and that this effect is eliminated when {alpha}1A function is blocked with the specific toxin agatoxin IVa or CdCl2, which suggests the discrete changes in channel function may contribute subtly to the disease phenotype (9), consistent with our present report of a gain of function mechanism.


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This work was supported by American Heart Association Grant AHA0335514N.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. S. Piedras-Rentería, Dept. of Physiology, Loyola Univ. Chicago, 2160 South First Ave., Maywood, IL 60153-5500 (e-mail: epiedra{at}lumc.edu)

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


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