Oral Presentation 2014 International Biophysics Congress

Model-based assessment of Cx43 hemichannel activity (#184)

Yeri Kim 1 , Liam M. Fisk 2 , Peter S. Freestone 3 , Colin R. Green 1
  1. Department of Ophthalmology, University of Auckland, Auckland, New Zealand
  2. Department of Mechanical Engineering, University of Canterbury, Christchurch, New Zealand
  3. Department of Physiology, University of Auckland, Auckland, New Zealand

Connexin 43 (Cx43) hemichannels are a subset of connexins that open in physiological and pathological conditions. Compared with other connexins, the biophysical properties of Cx43 hemichannels are not yet well characterised. We present a methodology for quantitatively selecting electrophysiology recordings suitable for model-based analysis of single Cx43 hemichannel activity. Human HeLa cells stably expressing a bicistronic Cx43IRESeGFP transcript were used for whole-cell patch clamp recordings. Single Cx43 hemichannel currents were evoked with voltage steps from 40 to 80 mV in 10 mV increments (30 s), in low extracellular Ca2+ solutions. Matlab was used to fit a Gaussian mixture model to the all-points histogram at each voltage step. The number of cells with 1/2/3 component Gaussians varied from 11/1/0 (n=12) at 40mV to 3/6/3 at 80mV (n=12). A component standard deviation (S.D.) much larger than the noise S.D indicates a low quality trace with excessive activity and/or macroscopic currents. The high quality traces at 80mV (n=6) had an average separation of 54.8 pS between distinct states. No distinct states were present in Cx43IRESeGFP HeLa cells treated with a generic connexin blocker LaCl3 (100 ┬ÁM; n=3) or in non-transfected HeLa cells (n=2) across all voltages. This study develops a quantitative method for accepting whole-cell patch clamp recordings to be used for single Cx43 hemichannel analysis. Our findings will provide the necessary groundwork for a model-based analysis of connexin mimetic peptide mechanisms of action.

Supported by University of Auckland Doctoral Scholarship to YK, and the Department of Ophthalmology