Gd3+ and calcium sensitive, sodium leak currents are features of weak membrane-glass seals in patch clamp recordings

Abstract

The properties of leaky patch currents in whole cell recording of HEK-293T cells were examined as a means to separate these control currents from expressed sodium and calcium leak channel currents from snail NALCN leak channels possessing both sodium (EKEE) and calcium (EEEE) selectivity filters. Leak currents were generated by the weakening of gigaohm patch seals by artificial membrane rupture using the ZAP function on the patch clamp amplifier. Surprisingly, we found that leak currents generated from the weakened membrane/glass seal can be surprisingly stable and exhibit behavior that is consistent with a sodium leak current derived from an expressible channel. Leaky patch currents differing by 10 fold in size were similarly reduced in size when external sodium ions were replaced with the large monovalent ion NMDG+. Leaky patch currents increased when external Ca2+ (1.2 mM) was lowered to 0.1 mM and were inhibited (>40% to >90%) with 10 µM Gd3+, 100 µM La3+, 1 mM Co2+ or 1 mM Cd2+. Leaky patch currents were relatively insensitive (<30%) to 1 mM Ni2+ and exhibited a variable amount of block with 1 mM verapamil and were insensitive to 100 µM mibefradil or 100 µM nifedipine. We hypothesize that the rapid changes in leak current size in response to changing external cations or drugs relates to their influences on the membrane seal adherence and the electro-osmotic flow of mobile cations channeling in crevices of a particular pore size in the interface between the negatively charged patch electrode and the lipid membrane. Observed sodium leak conductance currents in weak patch seals are reproducible between the electrode glass interface with cell membranes, artificial lipid or Sylgard rubber.

Figure 1. Ionic currents generated through a leaky patch (I_LP) generates a sodium leak conductance which is potentiated in low external calcium and is highly sensitive to Gd³⁺ block.

Left: Architecture of the cell attached patch as detailed by Fredrick Sachs . The membrane seal is the 10–50 micron of adherent membrane scaled along the inside walls of the glass pipette. The negatively charged glass and negatively-charged membrane generates a seal pocket that is filled with a lubricated layer of cations. The electro-osmotic flow of mobile cations in the seal space is the motor driving membrane patch creep. Right: Extracellular treatments that regulate whole cell leak currents in the patch seal. Less mobile cations like NMDG⁺ slows the inward electro-osmotic flow of ions compared to Na⁺ ions. Gd³⁺ has a high local charge density that titrates positive charges into the patch seal space, neutralizing the electrostatic repulsion of the negatively charged glass electrode and membrane, improving the seal resistance, and causing a decrease in leak current size. Lowering Ca²⁺ reduces the adhesive force between the glass and membrane and causes increases in leak current sizes.

Figure 2. Voltage ramp recordings of leaky patch current (I_LP) illustrating the stable sodium leak currents of differing sizes (approx. 100 pA, 200 pA, 400 pA, 800 pA) and their consistent behavior in successive sweeps (every 10 seconds) in response to perfusion.

(A) Representative tracings. Axes cross at zero. X = voltage (mV), Y = current (pA). (B) Size of outward currents were measured at +100 mV and the size of inward currents measured at −100 mV in the ramp protocol. There is a dramatic decrease in I_LP currents after perfusion of (1.2 mM [Ca]_ex, 150 mM [NMDG⁺]_ex), and an increase in I_LP currents after (0.1 mM [Ca]_ex, 150 mM [Na⁺]_ex), compared to perfusion of control external solution (1.2 mM [Ca]_ex, 150 mM [Na]_ex).

Figure 3. Percent change in current size of leaky patch current (I_LP) inward current (measured at −100 mV in ramp protocol) and I_LP outward current (measured at +100 mV in ramp protocol) in response to NMDG replacement of external Na ions or 0.1 mM Ca replacement of 1.2 mM external calcium.

Data points (left) and box plot (mean +/− SEM, right). Note that the relative change is remarkably consistent despite the dramatic difference in current densities of I_LP ranging from 0.38 to 171.28 pA/pF, with average 36.70+/−2.93 in 18 HEK-293T cells.

Figure 4. HEK-293T cell currents from a leaky patch (I_LP) have a characteristic drug blocking profile characteristic of ion channel currents.

Bath application of di- and trivalent ions, and calcium channel blockers and their block of I_LP during a 1 second voltage ramp. (A) Representative leaky patch currents. Axes cross at zero. X = voltage (mV), Y = current (pA). (B) Data points (left) and box plot (mean +/− SEM, right).