Supplementary Methods

Automated Patch Clamp Procedures

In preparation for a recording session, intracellular solutions (Table 1) were loaded into the intracellular compartments of the QPlate planar electrode. Cell suspensions were pipetted into the extracellular compartments of the QPlate planar electrode. After establishment of a whole-cell configuration, membrane currents were recorded using 48 parallel amplifiers in the Qpatch HT® system. Before digitization, the current records were low-pass filtered at one-fifth of the sampling frequency.

Valid whole-cell recordings should meet the following criteria:

(1) Membrane resistance (Rm)﹥200 MΩ

(2) Leak current ≤ 25% channel current.

Table 1 Intracellular solution composition*

Ion channel test system / Concentration (mM)
Cs-Asp. / MgCl2 / EGTA / Mg-ATP / Na2-ATP / Tris-GTP / HEPES
hCav1.2 / 130 / 5 / 10 / 0 / 4 / 0.1 / 10
hCav2.1 / 130 / 5 / 10 / 0 / 4 / 0.1 / 10
hCav2.2 / 130 / 5 / 10 / 0 / 4 / 0.1 / 10
hCav3.2 / 130 / 0 / 10 / 5 / 0 / 0.4 / 10

* The pH was adjusted to 7.2 with N-methyl-D-glucamine.

hCav1.2 test procedures

Onset and steady state block of hCav1.2/β2/α2δ channels were measured using a stimulus voltage pattern consisting of a depolarizing test pulse (duration, 200 ms; amplitude, 10 mV) at 10 s intervals from a -80 mV holding potential. Peak current was measured during the step to 10 mV. Leak current was measured after applying a saturating concentration of nifedipine (10μM, Sigma) at the end of each experiment to completely block current.

hCav2.1 test procedures

Onset and steady-state block of hCav2.1 currents were measured using a stimulus voltage pattern consisting of a depolarizing test pulse (depolarization 10 mV amplitude, 200 ms duration) repeated at 10 s intervals from a holding potential of -80 mV. Peak inward current amplitude was measured. Leak current was measured after applying a saturating concentration of cadmium (200 μM, Sigma) at the end of each experiment to completely block current.

hCav2.2 test procedures

Onset and steady-state block of hCav2.2 currents were measured using a pulse pattern with fixed amplitudes (depolarization: 10 mV amplitude, 200 ms duration) repeated at 10 s intervals from a holding potential of -80 mV. Peak inward current amplitude was measured. Leak current was measured after applying a saturating concentration of cadmium (200 μM, Sigma) at the end of each experiment to completely block current.

hCav3.2 test procedures

Onset and steady-state block of hCav3.2 currents were measured using a double pulse pattern consisting of a hyperpolarizing conditioning pulse (-120 mV amplitude, 250 ms duration) followed immediately by a depolarizing test pulse depolarization (-30 mV amplitude, 50 ms duration) from a -80 mV holding potential. The pulse pattern was repeated at 10 s intervals and peak test pulse current amplitude was measured.

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