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    L-Type Ca2+ Current Protocol

    Robert Harvey, Shailesh Agarwal

    Published: 2022-04-08 DOI: 10.17504/protocols.io.ba8mihu6
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    Abstract

    This protocol is for patch-clamp recordings of L-type Ca2+ current and slow delayed rectifier K+ current responses to norepinephrine and acetylcholine.

    Steps

    1.

    Membrane currents were recorded from isolated rat venticular myocytes using the whole-cell configuration of the patch clamp technique.

    2.

    Isolated cells were placed in a perfusion chamber (Warner Instruments) on the stage of an inverted microscope (Olympus IX71) and bathed in a K+-free extracellular solution containing (in mM) NaCl 137, CsCl 5.4, MgCl2 0.5, CaCl21.0, NaH2PO4 0.33, glucose 5.5, and HEPES 5 (pH 7.4), maintained at room temperature.

    3.

    Cells were patched using microelectrodes with resistances between 1 and 2 MΩ. Access resistance was compensated to obtain series resistance errors of less than 5 mV.

    4.

    Cells were dialyzed with a K+-free microelectrode solution containing (in mM): CsCl 130, TEA-Cl 20, EGTA 5, MgATP 5, TrisGTP 0.06, and HEPES 5 (pH 7.2).

    5.

    Whole cell currents were recorded under voltage-clamp conditions using a Multiclamp 700B voltage clamp amplifier, Digidata 1440A computer interface, and pClamp 11 data acquisition and analysis software (Molecular Devices). Data were lowpass filtered at 4 kHz, and sampled at 10 kHz.

    6.

    The membrane potential was held at -80 mV. A 50 ms pre-pulse to -40 mV was used to inactivate Na+channels. This was followed by a 100 ms test pulse to 0 mV to elicit the L‑type Ca2+ current.

    7.

    The time course of changes in the amplitude of the Ca2+ current was monitored by recording the amplitude of the peak inward current elicited during the test pulse to 0 mV applied once every 5 s.

    8.

    A stable baseline (about 5 minutes) was obtained before application of test drugs.

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