Selective T-type calcium channel block in thalamic neurons reveals channel redundancy and physiological impact of I(T)window

Abstract

Although it is well established that low-voltage-activated T-type Ca(2+) channels play a key role in many neurophysiological functions and pathological states, the lack of selective and potent antagonists has so far hampered a detailed analysis of the full impact these channels might have on single-cell and neuronal network excitability as well as on Ca(2+) homeostasis. Recently, a novel series of piperidine-based molecules has been shown to selectively block recombinant T-type but not high-voltage-activated (HVA) Ca(2+) channels and to affect a number of physiological and pathological T-type channel-dependent behaviors. Here we directly show that one of these compounds, 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2), exerts a specific, potent (IC(50) = 22 nm), and reversible inhibition of T-type Ca(2+) currents of thalamocortical and reticular thalamic neurons, without any action on HVA Ca(2+) currents, Na(+) currents, action potentials, and glutamatergic and GABAergic synaptic currents. Thus, under current-clamp conditions, the low-threshold Ca(2+) potential (LTCP)-dependent high-frequency burst firing of thalamic neurons is abolished by TTA-P2, whereas tonic firing remains unaltered. Using TTA-P2, we provide the first direct demonstration of the presence of a window component of Ca(2+) channels in neurons and its contribution to the resting membrane potential of thalamic neurons and to the Up state of their intrinsically generated slow (<1 Hz) oscillation. Moreover, we demonstrate that activation of only a small fraction of the T-type channel population is required to generate robust LTCPs, suggesting that LTCP-driven bursts of action potentials can be evoked at depolarized potentials where the vast majority of T-type channels are inactivated.

Figure 1.

TTA-P2 fully and reversibly blocks T-type Ca²⁺ current without affecting HVA Ca²⁺ currents in VB TC neurons. A, I_T were evoked every 20 s by step depolarization (200 ms long) to −50 mV after a 1 s hyperpolarizing prepulse to −100 mV. HVA Ca²⁺ currents were similarly evoked by step depolarization to 10 mV from the −60 mV holding potential. A1, Traces illustrate I_T (●, top traces) and HVA Ca²⁺ currents (○, bottom traces) recorded in the same VB neuron under control condition (a) and in the presence of 3 μm TTA-P2 (b, 2 min application; c, 5 min application). A2, The peak amplitude of I_T (●) and HVA Ca²⁺ currents (○) are plotted against time. TTA-P2 produced a 95% block of I_T without any effect on the HVA Ca²⁺ currents. B1, B2, Same protocols as in A1 and A2 in a different VB TC neuron. A full recovery of the amplitude of I_T was obtained after 55 min of wash-out of 1 μm TTA-P2. C, Dose–response curve of the effect of TTA-P2 on the amplitude of I_T. Data were fitted with the following equation: y = y_max/(1 + IC₅₀/x)ⁿ, where IC₅₀ = 22 nm, y_max = 94.5%, and n = 1.2. D, Same protocols as in A1 and A2. Interruption of the stimulating protocols during the first 8 min of 1 μm TTA-P2 application did not preclude the block of I_T. E, Activation and inactivation properties of I_T were estimated in the same neuron under control condition and after 20 min of 25 nm TTA-P2 application. The neuron was maintained at −60 mV between protocols. E1, I–V curves were constructed by successive step depolarizations from −80 to −45 mV (2.5 mV increments) preceded by a 1 s hyperpolarizing prepulse to −100 mV. Left traces illustrate currents evoked at the various potentials under control conditions (○) and in the presence of TTA-P2 (●). Despite the strong decrease in current amplitude induced by TTA-P2 application, the apparent voltage dependence of channel activation appears similar in control condition (○) and in the presence of TTA-P2 (●). E2, Normalized steady-state inactivation curves. Inactivation of T-type Ca²⁺ channels was induced using a 1 s prepulse of increasing potential (from −100 to −60 mV with 2.5 mV increments), and the resulting channel availability was estimated from the normalized current amplitude measured at −50 mV. Note the lack of any significant change in the presence of TTA-P2.

Figure 2.

Effect of TTA-P2 on I_Twindow of VB TC neurons. A1, The window T-type Ca²⁺ current evoked by a 10-s-long depolarizing voltage ramp from −100 to −40 mV preceded by a 1 s hyperpolarizing prepulse to −100 mV is fully blocked by 1 μm TTA-P2. A2, The voltage dependence of I_Twindow is presented. Note that the maximum amplitude of the current occurs around −63 mV.

Figure 3.

Effect of TTA-P2 on the resting membrane potential of TC neurons. A1, TTA-P2 (1 μm) induces a small hyperpolarization in a Wistar rat TC neuron held at −60 mV (top trace), concomitantly with the block of the LTCP evoked at the end of a hyperpolarizing step (bottom traces, with a–c marking the corresponding breaks in the top trace). A2, TTA-P2 (1 μm) does not hyperpolarize a TC neuron held at −70 mV (top trace) but still blocks the rebound LTCP (bottom traces, with a and b marking the corresponding breaks in the top trace). B1, TTA-P2 (1 μm) has no effect on the membrane potential of a TC neuron from a Cav3.1^−/− mouse recorded at −59 mV. Note the absence of a rebound LTCP after the hyperpolarizing steps and the lack of effect of TTA-P2 on the depolarizing sag of the hyperpolarizing response. B2, TTA-P2 (1 μm) induces a small hyperpolarization in a TC neuron from a wild-type mouse held at −61 mV (top trace), concomitantly with the block of the rebound LTCP (bottom traces, with a–c marking the corresponding breaks in the top trace).

Figure 4.

Effect of TTA-P2 on rat TC neuron excitability. A, Typical example of the reversible block by TTA-P2 (3-min-long application, 1 μm) of the rebound LTCP evoked at the end of a 1.5-s-long hyperpolarizing step in a rat TC neuron. Ba, The block of the rebound LTCP by 1 μm TTA-P2 was not associated with a change in the depolarizing sag (*) of the hyperpolarizing response to an intracellular current step. Bb, The membrane potential responses observed after repolarization in both conditions are superimposed at a larger time scale. Ca, Superimposed voltage traces illustrate the lack of effect of 1 μm TTA-P2 on membrane potential steps induced by injection of depolarizing currents (control, gray traces; TTA-P2, black traces). Cb, An I–V plot for similar experiments as in a from four TC neurons. Da, Typical example of the tonic firing observed in control condition and after 1 μm TTA-P2 application (same cell as in B). A similar number of action potentials (n = 32) is evoked during a 200 pA step depolarization in both control and TTA-P2 conditions. The first action potential recorded in control and in TTA-P2 conditions are enlarged in Db. Note the lack of effect of TTA-P2 on the threshold (Th), amplitude, half-width, and afterhyperpolarization (AHP) of the action potential. The graph in Dc presents the frequency–current plot from six neurons. CTR, Control.

Figure 5.

Effect of TTA-P2 on NRT neurons. A, Same protocol as in Figure 1A was used to record the I_T (●, top traces) and HVA Ca²⁺ currents (○, bottom traces) in an NRT neuron. TTA-P2 (1 μm) produced a 97% block of I_T without a concomitant decrease in the amplitude of the HVA Ca²⁺ current. B, Continuous voltage record from an NRT neuron showing that 1 μm TTA-P2 induces a small hyperpolarization concomitantly with the block of the rebound LTCP evoked by an hyperpolarizing step (breaks in the trace) before (a) and after a 4 and 8 min wash-in (b, c) of the antagonist. C, D, Same protocols as in Figure 4, B and D, were used to study the effect of 1 μm TTA-P2 on NRT neuron excitability. C, The block of the rebound LTCP by TTA-P2. E, The graph presents the frequency–current plot from nine neurons. CTR, Control.

Figure 6.

Effect of TTA-P2 on synaptically evoked LTCPs. A, Lack of effect of 1 μm TTA-P2 on evoked IPCSs (a; averages of 8 IPSCs) and EPSCs (b; averages of 7 EPSCs) recorded from two TC neurons. B, In a TC neuron maintained at −60 mV, rebound LTCPs were evoked by stimulations of the NRT at 20 s intervals in control condition and during successive applications of 20 and 50 nm TTA-P2. Ba, A concentration of 20 nm TTA-P2 had no effect on the high-frequency firing, whereas 50 nm TTA-P2 fully blocked the LTCP. However, as clearly seen on the superimposed traces in Bb, 20 nm TTA-P2 slowed the membrane repolarization and delayed the LTCP occurrence by 110 ms. C, In a TC neuron maintained at −70 mV, LTCPs were evoked by repetitive stimulations of the lemniscal pathway every 20 s under control conditions and while successively applying increasing concentrations of TTA-P2 (20, 50, and 100 nm and 1 μm). Note that a reduction in the LTCP-associated firing is observed with TTA-P2 concentrations ≥50 nm, which block >70% of the I_T (see Fig. 1C). The full block of the LTCP evoked by the sensory EPSP required a TTA-P2 concentration that abolishes I_T (i.e., 1 μm).

Figure 7.

Block of intrinsic thalamic oscillations by TTA-P2. A, Bath application of trans-ACPD (50 μm) produces a rapid depolarization of a cat MGB TC neuron resulting in continuous tonic firing of action potentials. Hyperpolarization of the cell by steady-state somatic current injection brings about the slow (<1 Hz) sleep oscillation (II). Application of TTA-P2 (3 μm) progressively decreases the duration of the slow oscillation Up state (III and IV) until it eventually resembles the delta oscillation (V), which also is abolished after a few additional minutes of drug exposure (VI). B, Superimposition of the Up states marked by the asterisks (*) in A shows the progressive reduction of their amplitude and duration during the continuous presence of TTA-P2. The inset shows a maximum-intensity z-series reconstruction of the Alexa Fluor 594-filled MGB neuron from which the data illustrated in A and B were collected. C, Reversible block by TTA-P2 of the slow and delta oscillations in a different cat MGB neuron. The break in the trace is 20 min.