- Calcium influx, required for keratinocyte differentiation, is enhanced by treatment with permeant cGMP. Moreover, the investigators have found a keratinocyte channel that is activated by cGMP. Finally, they have cloned complete and alternatively spliced isoforms of a cGMP channel that is similar to the calcium-permeable channel found in photoreceptor rod cells. The isoforms differ in their cGMP sensitivity, and their expression is unique in that it is developmentally regulated. The investigators hypothesize that this channel regulates keratinocyte differentiation through its role as cGMP-gated calcium influx pathway. The broad, long-term objective of these studies is to develop methods that modify keratinocyte differentiation by manipulating calcium influx into these cells. The health-relatedness of the project stems from the fact that abnormal keratinocyte differentiation underlies common skin diseases, both benign; psoriasis, abnormal wound healing; and malignant: squamous cell carcinoma. Thus, new approaches to modifying cell differentiation would have wide clinical applications.
The specific aims of the project are: 1) To correlate the exact proportion of each isoform with the degree of keratinocyte differentiation; 2) to compare channel characteristics in differentiating and undifferentiated keratinocytes; 3) to examine the channel's role in keratinocyte differentiation by overexpressing the native, alternatively spliced, or mutant isoforms in undifferentiated and differentiating keratinocytes. To accomplish these aims, the research design will first quantify the proportion of each isoform using the RNAse protection assay. Next, the functional difference conferred by the different isoforms will be examined by comparing 1) ion channel and whole cell current characteristics, using the patch clamp method; 2) calcium influx, using calcium-45 studies; and 3) intracellular calcium concentration, using Fluo-3 measurement. Finally, alteration of calcium influx and keratinocyte differentiation by overexpression of channel isoforms in keratinocytes will be tested.
