Mechanism of Electrical Rhythmicity in ICC

Project 1

Project Leader: Kenton M. Sanders, Ph.D.

Abstract

Interstitial cells of Cajal generate pacemaker activity in the GI tract that is responsible for peristaltic and segmental contractions in motility. Defects in pacemaker activity lead to disordered motility and unregulated transit of food and nutrients. Understanding how pacemaker cells work will provide new ideas about how to control GI motility and offer new suggestions about how to treat GI motility disorders.

Project 1 is investigating the mechanism of electrical rhythmicity in GI muscles. Phasic contractions are timed by (electrical slow waves that are generated by a specialized population of cells known as interstitial cells of Cajal (ICC). We developed a cell culture model of ICC during the previous funding period. These cells are spontaneously active, producing spontaneous transient inward currents (STICs), but the cells do not fully recapitulate electrical rhythmicity in intact muscle strips. For example, we could not activate large amplitude currents responsible for slow waves in these cells. Molecular studies showed that cultured ICC rapidly dedifferentiate and lose the ICC phenotype within a few days in culture. A fresh preparation of ICC was difficult to develop because ICC are difficult to identify in mixed populations of cells. In the next funding period we will utilize a new genetic tool we have created by engineering expression of a bright green fluorescent protein (copGFP) in ICC. Expression of the reporter makes it easy to find ICC in cell dispersions and to sort ICC by fluorescence activated cell sorting for molecular studies. Cells from these mice display spontaneous rhythmicity in the form of large amplitude spontaneous depolarizations that we believe are equivalent to slow waves in intact ICC networks. We will characterize the spontaneous activity of single ICC and describe the properties of the large amplitude 'autonomous' currents that underlie spontaneous depolarization. Autonomous currents can be pace by depolarization of single cells. We will explore the voltage-dependence of autonomous currents and determine the underlying voltage-sensor and mechanism that initiates these currents. We will also investigate how spontaneous activity in single ICC is regulated with the goal of understanding how slow wave frequency (and ultimately phasic contractile activity) is regulated. Several clinical studies have demonstrate loss of ICC in pathophysiological conditions, and this has resulted in hypotheses about the cause of GI motility disorders. We hypothesize that changes in ICC may precede the loss of cells, and we will characterize changes in ICC function during the development of type I1 diabetes. We will also study changes in ICC during the development of hyperplasia in pre-GIST ICC networks. New ideas about pacemaker activity in the gut will result from this work.