The effect of internal sodium and caesium on phasic contraction of patch-clamped rabbit ventricular myocytes.
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.
Levi
A J
AJ
Department of Physiology, School of Medical Sciences, University of Bristol, UK.
Mitcheson
J S
JS
Hancox
J C
JC
D000200:Action Potentials; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002586:Cesium / Q000378:metabolism*; D015314:Dialysis Solutions; D066298:In Vitro Techniques; D008297:Male; D009200:Myocardial Contraction / Q000502:physiology*; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000502:physiology; D012964:Sodium / Q000378:metabolism*; D013997:Time Factors; D016276:Ventricular Function
D015314:Dialysis Solutions; D002586:Cesium; D012964:Sodium; D011188:Potassium; D002118:Calcium