13
ZD 7288 concentration was reduced to 50 ∕√M in order to minimize potential cross over to
other ion channels.
2.3 Recordings
Recordings were made with an EPC-IO amplifier (HEKA, Lambrecht-Pfalz, Germany) in
either voltage clamp or current clamp mode. All power supply cables were shielded, which
along with the Faraday cage were well grounded [52]. Baseline noise levels recorded in volt-
age clamp mode with no pipette attached to the pipette holder were measured to be 140
fA RMS. Unless otherwise stated, data was low-pass filtered at 4 kHz with a Bessel filter,
and sampled at 10 kHz to avoid aliasing. Patchmaster software (HEKA, Lambrecht-Pfalz,
Germany) was used to generate stimulus pulses and record the data.
Glass electrodes were pulled from 1.5 mm OD thick-walled (0.86 mm ID) 10 cm glass
with filament (Sutter Instruments, Novato, CA) on a programmable P-97 puller (Sutter
Instruments, Novato, CA). Using thick glass reduced pipette capacitance and noise [52].
Smaller tipped pipettes were pulled for whole-cell recording in order to minimize the ef-
fects of wash-out. These pipettes measured 8-10 MΩ when filled with intracellular solu-
tion. These results are corrected for a calculated liquid junction potential of+14 mV [91].
Larger tipped pipettes were pulled for cell attached recordings in order to increase the num-
ber of HCN channels present in a patch. These pipettes measured between 1.5-3 MΩ when
filled with the cell attached solution. Giga-seals with these pipettes incorporated a large
area of membrane in order to maximize the HCN channel current. Typical cell attached