Clampfit version 10

Field excitatory postsynaptic potentials (fEPSPs) were recorded in the stratum radiatum of the hippocampal CA1 region by means of a glass microelectrode filled with 3 M NaCl (resistance 1–4 MΩ). The Schaffer collateral pathway was stimulated with concentric bipolar electrodes (Frederick Haer Co, Bowdoinham, ME, USA). After an optimal fEPSP wave was found, an input–output (I/O) curve was established with stimulating pulses (0.2 ms duration) at different intensities. Baseline fEPSP was recorded at 0.033 Hz with a stimulating strength adjusted to yield about 40% of the maximal response. After baseline responses had stably lasted for at least 30 min, LTP was induced by delivering theta burst stimuli (TBS; four trains of 10 bursts of four stimuli with 20 s, 200 ms, and 10 ms intervals between trains, bursts, and stimuli, respectively). The electrophysiological data was acquired with a multiclamp 700 A amplifier (Axon instruments, Molecular Devices, USA), filtered at 0.1–5 KHz, digitized at 10 KHz, and analyzed with Clampfit version 10.0 (Axon Instruments, USA). The analyzed data was further processed with Origin 5.1 (Microcal Software Northampton, MA).

In voltage-clamp experiments, Ca²⁺ currents were recorded using an extracellular solution containing 10 mM CaCl₂, 5 mM glucose, 120 mM tetraethylammonium chloride, 10 mM Hepes, 1 mM MgCl₂, 0.1 mM ethylene glycol-bis-b-aminoethyl ether-N,N,N',N'-tetraacetic acid (EGTA), pH 7.4. The patch pipette was filled with 130 mM CsCl, 1 mM EGTA, 0.5 mM MgCl₂, 10 mM Hepes, pH 7.4. During recordings, the bath contained 2 μM tetrodotoxin, to prevent Na⁺ current activation. The electrophysiological recordings were carried out at room temperature using the whole-cell configuration of the patch-clamp technique. Stimulation, acquisition, and data analysis were performed with the pCLAMP 9.0 and Clampfit version 10.0 software (Axon Instruments, Burlingame, USA). The linear components of leak and capacitive currents were canceled using the P/N4 method. The patch pipettes had resistances of 3–6 MΩ. To abolish the T-type contribution and select L-type Ca²⁺ currents, the cells were clamped from the holding potential of −90 to −30 mV for 750 ms, then a ramp was applied from −60 to +60 mV, with 10 mV increments and of 500 ms duration. A sampling interval of 50 kHz (20 μs/point) was used, and the currents were filtered at 2 kHz (Bessel filter).

The purified HsTMEM120A protein was reconstituted into the membrane of GUVs by using a mixture of cholesterol:DPhPC:azolectin (w:w:w) at a ratio of 1:5:17 or 1:10:15 with a protein:azolectin ratio of 1:85 (w/w) through a modified sucrose method (Battle et al., 2009 (link)). The bath and pipette solutions contained 500 mM NaCl, 10 mM CaCl₂, and 10 mM HEPES-NaOH (pH 7.4). Patch pipettes with resistances of 5–6 MΩ were used, and the patch resistance was increased to ~2 GΩ after the pipette was sealed tightly with the GUV membrane. A negative pressure was applied through a Suction Control Pro pump (Nanion) with a stepwise or linear protocol while data were recorded at a constant holding potential. The data were acquired at 50 kHz with a 0.5-kHz filter and a HumBug 50/60 Hz Noise Eliminator (Quest Scientific), using an EPC-10 amplifier (HEKA). The Clampfit Version 10.0 (Axon Instruments) was used for data analysis and Igor Pro 6.37A (WaveMetrics; RRID:SCR_000325) was used for making the graphs. The sample size represents the number of patches tested in the inside-out recordings. The number of patches tested was determined based on the observation of channel activities upon application of the negative pressure. Samples were grouped based on the proteins of interest reconstituted into the GUVs. The investigators were not blinded to group allocation.