Mutations in high-voltage-activated calcium channel genes stimulate low-voltage-activated currents in mouse thalamic relay neurons

Abstract

Ca2+ currents, especially those activated at low voltages (LVA), influence burst generation in thalamocortical circuitry and enhance the abnormal rhythmicity associated with absence epilepsy. Mutations in several genes for high-voltage-activated (HVA) Ca2+ channel subunits are linked to spike-wave seizure phenotypes in mice; however, none of these mutations are predicted to increase intrinsic membrane excitability or directly enhance LVA currents. We examined biophysical properties of both LVA and HVA Ca2+ currents in thalamic cells of tottering (tg; Cav2.1/alpha1A subunit), lethargic (lh; beta4 subunit), and stargazer (stg; gamma2 subunit) brain slices. We observed 46, 51, and 45% increases in peak current densities of LVA Ca2+ currents evoked at -50 mV from -110 mV in tg, lh, and stg mice, respectively, compared with wild type. The half-maximal voltages for steady-state inactivation of LVA currents were shifted in a depolarized direction by 7.5-13.5 mV in all three mutants, although no alterations in the time-constant for recovery from inactivation of LVA currents were found. HVA peak current densities in tg and stg were increased by 22 and 45%, respectively, and a 5 mV depolarizing shift of the activation curve was observed in lh. Despite elevated LVA amplitudes, no alterations in mRNA expression of the genes mediating T-type subunits, Cav3.1/alpha1G, Cav3.2/alpha1H, or Cav3.3/alpha1I, were detected in the three mutants. Our data demonstrate that mutation of Cav2.1 or regulatory subunit genes increases intrinsic membrane excitability in thalamic neurons by potentiating LVA Ca2+ currents. These alterations increase the probability for abnormal thalamocortical synchronization and absence epilepsy in tg, lh, and stg mice.

Fig. 1.

Increased LVA Ca²⁺ peak current in tottering, lethargic, andstargazer. A, Biocytin-filled thalamic neuron in the LDN of thalamus from wild-type mouse (C57BL/6J).B, Representative LVA current traces from TCs of the LDN in control, tottering, lethargic, andstargazer mice. The cell capacitance values of these four neurons were 101.25, 107.1, 95.5, and 95.34 pF, respectively. Holding potential, −70 mV. The membrane potential was prepulsed to −110 mV for 3 sec before stepping to −50 mV for 200 msec. Decay of the current was fitted by a single-exponential function (dotted line). No significant alterations in macroscopic current decay were found. The representative time constants τ for decay were 29.2, 29.7, 30.1, and 31.8 msec, respectively, in control, tg,lh, and stg mice. C, Elevated LVA Ca²⁺ current amplitude and peak current density from mutant TCs. LVA currents were evoked at the same membrane potential as described in B. The mean current amplitude and peak current densities were −926.3 ± 182.2 pA and 9.5 ± 1.3 pA/pF in control, 1514.9 ± 142 pA and 17.4 ± 1.2 pA/pF in stg, −1571.9 ± 106.8 pA and 17.63 ± 1.6 pA/pF in tg, and −1852.1 ± 118.9 pA and 19.6 ± 1.0 pA/pF in lh mice. *p < 0.05; **p < 0.01 versus control.

Fig. 2.

Depolarized shift of the voltage dependence of LVA calcium channel availability (steady-state inactivation) intottering, lethargic, andstargazer mutants. A, Representative current traces for SSI of LVA Ca²⁺ currents. A standard double-pulse protocol for steady-state inactivation was given from the holding potential of −70 mV. A 4 sec prepulse at potentials ranging from −120 to −40 mV preceded each depolarization, followed by a subsequent voltage step to −50 mV for 200 msec. The interpulse interval was 10 sec. B, Normalized current–voltage curves for SSI of LVA Ca²⁺ currents. Current amplitude from the inactivation protocol, normalized to maximum, was plotted as a function of prepulse membrane potentials and best fitted with a Boltzmann function: I/I_max = {1 + exp(V −V_1/2)/k} − 1. The pooled half-maximal voltages (V_1/2) and slopes (k) were −92.3 ± 0.16 and 6.8 ± 0.16 mV in control, −84.8 ± 0.17* and 6.51 ± 0.15 mV intg, −78.72 ± 0.3** and 6.0 ± 0.27 mV inlh, and −78.6 ± 0.3 ** and 6.0 ± 0.27 mV instg, respectively. *p < 0.05; **p < 0.01.

Fig. 3.

Recovery from inactivation of LVA Ca²⁺ currents. A, Representative current traces for recovery from inactivation of LVA currents in control, tg, lh, and stg. The holding potential was set to −50 mV, and 50 mV hyperpolarizations of incremental duration were applied. LVA peak amplitude was measured after returning to −50 mV. B, Recovery from inactivation curves. Recovery curves were established by plotting the normalized peak amplitude versus duration. The recovery curves followed a two-exponential time course, best fitted with fast time constant (τ₁) of 230, 240, 196, and 225 msec for control,tg, lh, and stg, and slow time constant (τ₂) of 1300, 1250, 1100, and 1270 msec for control, tg, lh, andstg, respectively.

Fig. 4.

Representative superimposed HVA Ca²⁺ current traces from thalamocortical cells in control, tg, lh, and stgmice. The I–V protocol consisted of a 3 sec prepulse potential at −60 mV, followed by voltage steps (200 msec) ranging from −80 to +60 mV in 5 mV increments, with the holding potential maintained at −70 mV.

Fig. 5.

Peak HVA Ca²⁺ currents are increased in tottering and stargazer but not lethargic mice. A, Current density–voltage curves for HVA Ca²⁺ currents, constructed by plotting the normalized current amplitude at various membrane potentials. The voltage protocol used was identical to that described in Figure 4. B, Peak current density and current amplitude from A. The mean peak current density was 10.12 ± 1.2, 13.03 ± 0.6, 9.42 ± 0.4, and 17.99 ± 2.1 pA/pF in control, tg,lh, and stg (**p < 0.01 vs control). The mean current amplitude was −885.51 ± 112.8, 1204.21 ± 86.4, −901.3 ± 48.7, and −1567.6 ± 188 pA in control, tg, lh, andstg mutants (**p < 0.01 vs control). C, SSA of HVA Ca²⁺ currents in control, tg, lh, andstg mice. The steady-state conductance (G) and voltage (V) data were transformed from I–V data shown inA. The solid and dotted curves are fits of the data to the Boltzmann equation of the following form: G/G_max = 1/(1 + exp(V_1/2 −V)/k), whereG_max is maximum conductance,V_1/2 is half-maximal voltage, andk is the slope. The mean values ofV_1/2 and slope for SSA of HVA currents are −22.0 ± 0.17 and 5.5 ± 0.15 mV in control, −21.7 ± 0.16 and 4.5 ± 0.14 mV in tg, −17.0 ± 0.11* and 4.7 ± 0.1 mV in lh mice, and −23.9 ± 0.18 and 4.6 ± 0.16 mV in stg, respectively (*p < 0.05 vs control).

Fig. 6.

Expression of T-type calcium channel genes in tg, lh, and stgthalamus. The three genes, Cacna1g(Cav3.1/α1G),Cacna1h (Cav3.2/α1H), andCacna1i (Cav3.3/α1I) were expressed in distinct and primarily non-overlapping patterns in coronal sections of mouse brain. Cav3.1 mRNA was detected at highest levels in neocortex and thalamus and in hippocampal dentate granule cells, with lower levels in hippocampal CA1–CA2 regions. Cav3.2 expression appeared to be restricted to hippocampal dentate gyrus and CA1–CA3 regions in the sections shown above. Cav3.3 expression was the most broadly distributed of the three T-type genes in mouse brain, with high levels of mRNA observed in nRT, habenula, hippocampal CA1 region, neocortex, and striatum. These patterns were not appreciably altered in homozygous tg,lh, or stg mutant mice.