Sedentary keratinocytes at the edge of a skin wound migrate into the wound, guided by the generation of an endogenous electric field (EF) generated by the collapse of the transepithelial potential. The center of the wound quickly becomes more negative than the surrounding tissue and remains the cathode of the endogenous EF until the wound is completely re-epithelialized. This endogenous guidance cue can be studied in vitro. When placed in a direct current (DC) EF of physiological strength, 100 V/m, keratinocytes migrate directionally toward the cathode in a process known as galvanotaxis. Although a number of membrane-bound (e.g., epidermal growth factor receptor (EGFR), integrins) and cytosolic proteins (cAMP, ERK, PI3K) are known to play a role in the downstream signaling mechanisms underpinning galvanotaxis, the initial sensing mechanism for this response is not understood. To investigate the EF sensor, we studied the migration of keratinocytes in a DC EF of 100 V/m, alternating current (AC) EFs of 40 V/m at either 1.6 or 160 Hz, and combinations of DC and AC EFs. In the AC EFs alone, keratinocytes migrated randomly. The 1.6 Hz AC EF combined with the DC EF suppressed the direction of migration but had no effect on speed. In contrast, the 160 Hz AC EF combined with the DC EF did not affect the direction of migration but increased the migration speed compared to the DC EF alone. These results can be understood in terms of an electromechanical transduction model, but not an electrodiffusion/osmosis or a voltage-gated channel model.

Repair to damaged tissue requires directional cell migration to heal the wound. Immediately upon wounding an electrical guidance cue is created with the cathode of the electric field (EF) located at the center of the wound. Previous research has demonstrated directional migration of keratinocytes toward the cathode when an EF of physiological strength (100-150 mV/mm) is applied in vitro, but the "sensor" by which keratinocytes sense the EF remains elusive. Here we use a customized chamber design to facilitate the application of a direct current (DC) EF of physiological strength (100 mV/mm) to keratinocytes whilst pharmacologically modulating the activation of both connexin hemichannels and purinergic receptors to determine their role in EF-mediated directional keratinocyte migration, galvanotaxis. In addition, keratinocytes were exposed to DiSCAC2 (3) dye to visualize membrane potential changes within the cell upon exposure to the applied DC EF. Here we unveil ATP-medicated mechanisms that underpin the initiation of keratinocyte galvanotaxis. The application of a DC EF of 100 mV/mm releases ATP via hemichannels activating a subset of purinergic P2 Y receptors, locally, to initiate the directional migration of keratinocytes toward the cathode in vitro, the center of the wound in vivo. The delineation of the mechanisms underpinning galvanotaxis extends our understanding of this endogenous cue and will facilitate the optimization and wider use of EF devices for chronic wound treatment. J. Cell. Physiol. 230: 181-191, 2016. © 2015 Wiley Periodicals, Inc.