Mechanisms of Mechanotransduction in Merkel cells
Merkel cell-neurite complexes are highly sensitive touch receptors comprising sensory afferents and epidermal Merkel cells. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To ask whether Merkel cells are genetically programmed to be excitable cells, we purified Merkel cells from touch domes and used DNA microarrays to compare gene expression in Merkel cells and other epidermal cells. We identified 362 Merkel-cell-enriched transcripts including neuronal transcription factors, presynaptic molecules and ion-channel subunits. Antibody staining of skin sections showed that Merkel cells are immunoreactive for presynaptic proteins, including piccolo, Rab3C, VGLUT2 and cholecystokinin. These data indicate that Merkel cells are poised to release glutamate and neuropeptides. Finally, using Ca2+ imaging, we discovered that Merkel cells have L-type and P/Q-type voltage-gated Ca2+ channels, which have been shown to trigger vesicle release at synapses.
We also asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and direct indentation with probes while responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in actin-filled processes. Third, voltage activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ induced Ca2+ release amplifies signals from swelling activated cation channels. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of Merkel-cell hypotonic responses. Collectively, these results directly demonstrate that Merkel cells are mechanosensitive, identify cellular signaling mechanisms that mediate mechanically evoked responses, and support the hypothesis that Merkel cells contribute to touch reception in the Merkel cell-neurite complex.