Biophysically Based Modeling of the Interstitial Cells of Cajal: Current Status and Future Perspectives

In electrically nonexcitable cells, Ca(2+) influx is essential for regulating a host of kinetically distinct processes involving exocytosis, enzyme control, gene regulation, cell growth and proliferation, and apoptosis. The major Ca(2+) entry pathway in these cells is the store-operated one, in which the emptying of intracellular Ca(2+) stores activates Ca(2+) influx (store-operated Ca(2+) entry, or capacitative Ca(2+) entry). Several biophysically distinct store-operated currents have been reported, but the best characterized is the Ca(2+) release-activated Ca(2+) current, I(CRAC). Although it was initially considered to function only in nonexcitable cells, growing evidence now points towards a central role for I(CRAC)-like currents in excitable cells too. In spite of intense research, the signal that relays the store Ca(2+) content to CRAC channels in the plasma membrane, as well as the molecular identity of the Ca(2+) sensor within the stores, remains elusive. Resolution of these issues would be greatly helped by the identification of the CRAC channel gene. In some systems, evidence suggests that store-operated channels might be related to TRP homologs, although no consensus has yet been reached. Better understood are mechanisms that inactivate store-operated entry and hence control the overall duration of Ca(2+) entry. Recent work has revealed a central role for mitochondria in the regulation of I(CRAC), and this is particularly prominent under physiological conditions. I(CRAC) therefore represents a dynamic interplay between endoplasmic reticulum, mitochondria, and plasma membrane. In this review, we describe the key electrophysiological features of I(CRAC) and other store-operated Ca(2+) currents and how they are regulated, and we consider recent advances that have shed insight into the molecular mechanisms involved in this ubiquitous and vital Ca(2+) entry pathway.

Calcium (Ca(2+))-activated chloride channels are fundamental mediators in numerous physiological processes including transepithelial secretion, cardiac and neuronal excitation, sensory transduction, smooth muscle contraction and fertilization. Despite their physiological importance, their molecular identity has remained largely unknown. Here we show that transmembrane protein 16A (TMEM16A, which we also call anoctamin 1 (ANO1)) is a bona fide Ca(2+)-activated chloride channel that is activated by intracellular Ca(2+) and Ca(2+)-mobilizing stimuli. With eight putative transmembrane domains and no apparent similarity to previously characterized channels, ANO1 defines a new family of ionic channels. The biophysical properties as well as the pharmacological profile of ANO1 are in full agreement with native Ca(2+)-activated chloride currents. ANO1 is expressed in various secretory epithelia, the retina and sensory neurons. Furthermore, knockdown of mouse Ano1 markedly reduced native Ca(2+)-activated chloride currents as well as saliva production in mice. We conclude that ANO1 is a candidate Ca(2+)-activated chloride channel that mediates receptor-activated chloride currents in diverse physiological processes.

Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.