The effect of internal sodium and caesium on phasic contraction of patch‐clamped rabbit ventricular myocytes.
Authors: Levi A J , Mitcheson J S , Hancox J C 
Published: December 2014

1. The voltage dependence of phasic contraction was assessed in rabbit ventricular myocytes. Phasic contraction at all potentials was abolished by exposure to ryanodine‐thapsigargin, showing that it was due primarily to Ca2+ release from the sarcoplasmic reticulum (SR). Experiments were performed at 35 degrees C, cells were whole‐cell patch clamped and contraction was measured optically as unloaded shortening. Cells were held at ‐40 mV to inactivate the Na+ current (INa) and T‐type Ca2+ current. A standard cellular Ca2+ load was established by applying a train of conditioning pulses at 0.5 Hz before each test pulse. The effect of replacing K+ with Cs+ in the dialysing pipette solution, and the effect of altering dialysing [Na+] between 0 and 20 mM, was assessed on contraction. 2. Cells dialysed with a K(+)‐based, Na(+)‐free solution exhibited a ‘bell‐shaped’ voltage dependence of the L‐type Ca2+ channel current (ICa,L), with a maximum ICa,L at +10 mV. Replacing internal K+ with Cs+, or altering pipette [Na+], did not affect the voltage dependence of ICa,L. 3. The voltage dependence of phasic contraction in cells dialysed with a K(+)‐based solution was modulated by pipette [Na+]. The voltage dependence of phasic contraction was bell‐shaped with 0 Na+, became much loss bell‐shaped with 10 mM Na+ and with 20 mM Na+ the phasic contraction elicited at +100 mV was 1.6‐fold larger than that at +10 mV. 4. Replacing 80% of K+ with Cs+ in the pipette dialysis solution led to a significant reduction in contraction amplitude and a more rapid decline in contraction amplitude after beginning the dialysis of the cell. 5. Cells dialysed with a Cs(+)‐based solution displayed a voltage dependence of phasic contraction which was more bell‐shaped (i.e. more similar to that of ICa,L) than that obtained with the corresponding K(+)‐based dialysis solution. The level of pipette [Na+] still modulated the voltage dependence of phasic contraction in cells dialysed with a Cs(+)‐based solution. 6. Time‐to‐peak contraction (tpk) also displayed voltage dependence; it had a minimum value between 0 and +20 mV (the voltage range for maximum ICa,L), but increased at more negative and positive potentials. Alteration of tpk contraction is discussed in relation to the stochastic behaviour of L‐type Ca2+ channels and SR Ca2+ release channels. 7. The shape of the voltage dependence of contraction in rabbit myocytes at 35 degrees C is modulated by dialysing [Na+] over the tested range, 0‐20 mM. Modulation of voltage dependence of contraction by dialysing [Na+] is consistent with an influence of reverse Na(+)‐Ca2+ exchange in triggering intracellular Ca2+ release, in addition to the trigger Ca2+ which enters via ICa,L. 8. The marked effect of dialysing Cs+ on contraction amplitude, and on the voltage dependence of phasic contraction, does not appear to have been reported previously. Internal dialysis with Cs+ is a commonly used technique for blocking interfering outward K+ currents, in order to measure ICa,L more selectively. The present study suggests that Cs+ might also interfere with processes involved in excitation‐contraction coupling and indicates that it might be wise to exercise caution with the use of internal Cs+ in experiments investigating excitation‐contraction coupling.