Altered calcium channel currents in Purkinje cells of the neurological mutant mouse leaner

Abstract

Mutations of the alpha1A calcium channel subunit have been shown to cause such human neurological diseases as familial hemiplegic migraine, episodic ataxia-2, and spinocerebellar ataxia 6 and also to cause the murine neurological phenotypes of tottering and leaner. The leaner phenotype is recessive and characterized by ataxia with cortical spike and wave discharges (similar to absence epilepsy in humans) and a gradual degeneration of cerebellar Purkinje and granule cells. The mutation responsible is a single-base substitution that produces truncation of the normal open reading frame beyond repeat IV and expression of a novel C-terminal sequence. Here, we have used whole-cell recordings to determine whether the leaner mutation alters calcium channel currents in cerebellar Purkinje cells, both because these cells are profoundly affected in leaner mice and because they normally express high levels of alpha1A. In Purkinje cells from normal mice, 82% of the whole-cell current was blocked by 100 nM omega-agatoxin-IVA. In Purkinje cells from homozygous leaner mice, this omega-agatoxin-IVA-sensitive current was 65% smaller than in control cells. Although attenuated, the omega-agatoxin-IVA-sensitive current in homozygous leaner cells had properties indistinguishable from that of normal Purkinje neurons. Additionally, the omega-agatoxin-IVA-insensitive current was unaffected in homozygous leaner mice. Thus, the leaner mutation selectively reduces P-type currents in Purkinje cells, and the alpha1A subunit and P-type current appear to be essential for normal cerebellar function.

Fig. 1.

Barium currents in Purkinje cells of wild-type and leaner mice. A1–A3, Inward currents elicited by depolarizing voltage steps from a holding potential of −80 mV in wild-type (A1),tg^la/+ (A2), andtg^la/tg^la(A3) cells. Note the difference in current densities between cell types (I_pk for wild-type = −134.3 pA/pF, tg^la/+ = −132.7 pA/pF, andtg^la/tg^la = −70.4 pA/pF). B1–B3, Conductance versus voltage curves plotted from currents in A1–A3. The data were fitted with a single Boltzmann function. The G–V curves had similar half-activation voltages (for wild-type cell,V_1/2 = −25.3 mV;tg^la/+ cell,V_1/2 = −23.9 mV;tg^la/tg^lacell, V_1/2 = −23.2 mV). The slope factor of thetg^la/tg^lacurve (6.2) was greater than that for thetg^la/+ (5.7) and wild-type cells (3.9).

Fig. 2.

Inactivation of barium currents in wild-type andleaner cells. A1–A3, Currents elicited by a two-pulse protocol: 2 sec depolarizing prepulses (−110 to 30 mV) followed by a test pulse to −10 mV with a 10 sec time interval between measurements. Peak current density during the test pulse was used to plot inactivation curves (I/I_max vs prepulse voltage; data were fit with a single Boltzmann function). The half-inactivation voltages of the inactivation curves were −46.2 mV (slope factor, 18.8) for the wild-type cell (A1), −52.3 mV (slope factor, 18.4) for the tg^la/+ cell (A2), and −50.7 mV (slope factor, 19.1) for thetg^la/tg^lacell (A3). B1–B3, Barium currents elicited by a long 2 sec depolarizing voltage step to −10 mV. Percent inactivation was 69% in the wild-type cell (B1), 55% in the tg^la/+ cell (B2), and 46% in thetg^la/tg^lacell (B3). The same cells were used in Aand B.

Fig. 3.

Effects of ω-AgTx-IVA on barium currents in wild-type and leaner Purkinje cells. A1, A2, Barium currents before and after application of 100 nm ω-AgTx-IVA. ω-AgTx-IVA blocked 82% of the current in the tg^la/+ cell (A1) and 63% in thetg^la/tg^lacell (A2). Note the similarities of the currents resistant to ω-AgTx-IVA in A1 and A2. The current densities of the ω-AgTx-IVA-resistant current were −24.2 pA/pF for the tg^la/+ cell and −25.2 pA/pF for thetg^la/tg^lacell. The current remaining after ω-AgTx-IVA block represents non-P-type currents. B1, B2, ω-AgTx-IVA-sensitive currents were derived by subtracting the control and ω-AgTx-IVA traces in A1 and A2. The ω-AgTx-IVA-sensitive current from thetg^la/+ cell had a density of −107.1 pA/pF (B1), and that from thetg^la/tg^lacell was −37.5 pA/pF (B2). C, Plot of the peak current density versus time for the cells in A1and A2. Gray circles represent data from the tg^la/+ cell in A1, and the black circles were from thetg^la/tg^lacell in A2. D, ω-AgTx-IVA-sensitive currents from B1 and B2 were scaled and superimposed.

Fig. 4.

The bar graph depicts the current density of the barium currents in wild-type and leaner Purkinje cells. The total barium current densities intg^la/tg^lacells were reduced compared with tg^la/+ cells (gray bars; p < 0.01). There was no significant difference between the current densities of the ω-AgTx-IVA-resistant currents (the current remaining after ω-AgTx-IVA block; black bars) intg^la/+ andtg^la/tg^lacells. The mean current density of the ω-AgTx-IVA-sensitive current (striped bars) intg^la/tg^lacells was 65% smaller than that intg^la/+ cells (p≪ 0.0001).

Fig. 5.

Activation of ω-AgTx-IVA-sensitive currents in tg^la/+ andtg^la/tg^lacells. A1, A2, Barium currents elicited by depolarizing voltage steps from a −80 mV holding potential. ω-AgTx-IVA-sensitive currents were derived by subtracting currents recorded after ω-AgTx-IVA block from control currents. Current density was markedly attenuated in thetg^la/tg^lacell (I_pk = −17.3 pA/pF) compared with thetg^la/+ cell (I_pk = −121.3 pA/pF). B1, B2, G–V curves for the ω-AgTx-IVA-sensitive currents in A1 and A2. Voltage dependence of activation was similar for the tg^la/+ currents (V_1/2 = −21.9 mV; k = 5.0) and thetg^la/tg^lacurrents (V_1/2 = −26.6 mV; k = 4.7).

Fig. 6.

Inactivation properties of ω-AgTx-IVA-sensitive currents in tg^la/+ andtg^la/tg^lacells. A1, A2, Voltage dependence of inactivation was described using a Boltzmann function. Thetg^la/+ currents exhibited a half-inactivation voltage of −41.2 mV (k = 13.2), and thetg^la/tg^lacurrents were half-maximally inactivated at −55.6 mV (k = 17.4).B1, B2, Percent inactivation was 51% for thetg^la/+ current and 67% for thetg^la/tg^lacurrent.