Electrophysiological characterization of human keratinocytes using the patch-clamp technique.

Abstract

The living, not-cornified part of the epidermis consists mainly of keratinocytes. The control mechanisms of proliferation and differentiation are only partly understood. Similarly, the influence of ionic channels of the cell membrane on the proliferation of keratinocytes remains unclear. Preliminary investigations point to a relation between the opening of ionic channels and keratinocyte proliferation. Therefore, voltage clamp experiments were performed to gain further knowledge of the electrophysiological characteristics of human keratinocyte cellular membranes. In-vitro cultured keratinocytes of the cell line HaCaT were characterized by means of the voltage-clamp technique. As measured in the whole-cell configuration, changing the extracellular K+ or Cl--concentration shifted the membrane potential of HaCaT cells. Application of the patch-clamp-technique in the cell-attached and inside-out configuration revealed an ionic channel with a conductance of multiples of 200 pS. The reversal potential of the single channel current was shifted by substituting of intracellular Cl- by aspartate-. Channel openings disappeared after addition of 0.1 mM of the anion channel blocker 4',4' diisothiocyanato-stilbene-2-2' disulfonic acid (DIDS). It is concluded that this channel contributes to the Cl--conductance of the cellular membrane and is a determinant of the membrane potential of human keratinocytes. This channel may represent a target for pharmacological manipulation of the membrane potential and possibly the growth of human keratinocytes in dermatological proliferation disorders.