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

PLoS One. 2014 Jun 19;9(6):e98808. doi: 10.1371/journal.pone.0098808. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium / metabolism*
  • Calcium Channels / biosynthesis
  • Calcium Channels / metabolism
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism*
  • HEK293 Cells
  • Humans
  • Nifedipine / pharmacology
  • Patch-Clamp Techniques
  • Sodium / metabolism*
  • Sodium Channels / biosynthesis*
  • Sodium Channels / metabolism
  • Verapamil / administration & dosage

Substances

  • Calcium Channels
  • NALCN protein, human
  • Sodium Channels
  • Sodium
  • Verapamil
  • Nifedipine
  • Calcium

Grant support

The authors would like to thank the Heart and Stroke Foundation and NSERC Discovery Operating for operating grant funding to JDS. The authors thank an NSERC-MSFSS award for providing Adriano Senatore an opportunity to examine the functions of NALCN with the CNRS group including Dr. Monteil in Montpellier, France. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.