Ionic diffusion in voltage-clamped isolated cardiac myocytes. Implications for Na,K-pump studies.

Area of Science:

• Cardiology
• Cell Physiology
• Biophysics

Background:

• The whole-cell voltage-clamp technique is crucial for electrophysiological studies.
• It assumes electrode solution composition influences intracellular ion concentrations, particularly for Na,K-pump research.
• The direct relationship between electrode and intracellular ion concentrations, especially sodium, remains unconfirmed.

Purpose of the Study:

• To investigate if the sodium concentration in the pipette electrode ([Na+]pip) can set and clamp the intracellular sodium concentration at the Na,K-pump's binding sites ([Na+]ps).
• To establish a method for precisely defining the Na,K-pump's dependence on intracellular sodium levels.

Main Methods:

• Developed a 3D mathematical model of ionic diffusion in whole-cell patch-clamped myocytes.
• Simulated the effects of experimental parameters on intracellular sodium concentration ([Na+]ps).
• Experimentally validated model predictions using voltage-clamped isolated ventricular myocytes and measuring Na current reversal potential.

Main Results:

• Mathematical simulations showed that steady-state [Na+]ps is sensitive to electrode pore size, cell length, and Na+ pumping rate.
• At steady state, simulated [Na+]ps varied by 5% or less from [Na+]pip, depending on the pump rate.
• Experimental data supported the model, indicating that electrode [Na+] can regulate steady-state intracellular [Na+]ps.

Conclusions:

• Steady-state intracellular sodium concentration at the Na,K-pump binding sites ([Na+]ps) can be effectively regulated by the sodium concentration in suction pipette electrodes ([Na+]pip).
• This finding validates the use of whole-cell suction micro-electrodes for assessing the Na,K-pump's dependence on intracellular sodium in isolated cardiac myocytes.
• The study provides a reliable method for future research on cardiac electrophysiology and ion transport mechanisms.