In the last decade, a number of studies have demonstrated the involvement of cytoskeletal proteins in secretion, and some studies implicate direct interaction of cytoskeleton protein with SNAREs [2, 13- 17]. Furthermore, actin and microtubule-based cytoskeleton have been implicated in intracellular vesicle traffic [2, 15]. Fodrin, which was previously implicated in exocytosis , has recently been shown to directly interact with SNAREs . Studies demonstrate α-fodrin to regulate exocytosis via its interaction with t-SNARE syntaxin family of proteins . The c-terminal coiled coil region of syntaxin interacts with α-fodrin, a major component of the submembranous cytoskeleton. Similarly, vimentin filaments interact with SNAP23/25 and hence are able to control the availability of free SNAP23/25 for assembly of the SNARE complex . All these findings suggested that vimentin, α- fodrin, actin, and SNAREs may be part of the porosome complex. Additional proteins such as v- SNARE (VAMP or synaptobrevin), synaptophysin and myosin, may associate when the porosome establishes continuity with the secretory vesicle membrane. The globular tail domain of myosin V contains binding site for VAMP, which is bound in a calcium independent manner . Further interaction of myosin V with syntaxin requires both calcium and calmodulin. It has been suggested that VAMP acts as a myosin V receptor on secretory vesicles and regulates formation of the SNARE complex . Interaction of VAMP with synapto- physin and myosin V has also been observed . In agreement with earlier findings in other tissues, our studies have demonstrated the association of SNAP- 23, syntaxin 2, cytoskeletal proteins actin, α-fodrin, and vimentin, and calcium channels β3 and α1c, together with the SNARE regulatory protein NSF, in porosomes [8, 11] (Fig. 10). Additionally, chloride ion channels ClC2 and ClC3 were also identified as part of the porosome complex . Isoforms of the various proteins identified in the porosome complex, were subsequently demonstrated using 2D-BAC gels electrophoresis . Three isoforms each of the calcium ion channel and vimentin were clearly identifiable. Although multiple spots were identified in several of the immunoblots, the low molecular weight spots may represent proteolytic degredation of the parent molecule . Using yeast 2-hybrid analysis, our study confirms the presence and further reveals the interactions of some of these proteins with t-SNAREs within the porosome complex (unpublished). The size and shape of the immunoisolated porosome complex when examined using both negative staining electron microscopy and AFM, was revealed in greater detail. The images of the immunoisolated porosome obtained by both EM and AFM were super-imposable (Fig. 11) . To further test whether the immunoisolated supramolecular complex was indeed the porosome, the complex was reconstituted into artificial liposomes, and the liposome-reconstituted complex examined using TEM (Fig. 12) . Transmission electron micrographs reveal a 150-200-nm cupshaped basket-like structure as observed of the porosome when co-isolated with ZGs. The important question then remained, are such reconstituted porosomes functional? To answer this question, the complex was also reconstituted into lipid membranes and challenged with isolated secretory vesicles (ZGs) in an electrophysiological bilayer setup (EPC9). Both the electrical activity of the reconstituted membrane as well as the transport of vesicular contents from the cis to the trans compartment, was monitored in the EPC9 electrophysiologicallipid membrane setup. Results of these experiments demonstrate that the lipid membrane-reconstituted porosomes are functional supramolecular complexes (Fig. 13) . When the supramolecular porosome complexes is reconstituted into the lipid bilayer membrane in the EPC9 setup (Fig. 13A), ZG fused with the bilayer as demonstrated by an increase in capacitance and conductance, and a time-dependent release of amylase (one of the major contents of ZGs) from cis to the trans compartment of the chamber. Amylase is detected using immunoblot analysis of the buffer in the cis and trans chambers (Fig. 13B), using a previously characterized amylase specific antibody . As observed in immunoblot assays of isolated porosomes (Fig. 10), chloride channel activities is additionally demonstrated within the reconstituted supramolecular porosome complex (Fig. 13C). Further, the chloride channel inhibitor DIDS, was found to inhibit current activity in the porosomereconstituted bilayer. Contrarily, although our immunoblot analysis of porosomes demonstrate the association of calcium channels with the complex, we were unable to detect calcium channel activities in the reconstituted membrane. This may have been due to inactivation of the associated calcium channels in the complex as a result of low pH wash during immunoisolation, since recently we have been able to recover this activity (unpublished). The role of the chloride channel in the porosome complex remains unknown at this time. In summary, these studies demonstrate that the porosome is a 100-150- nm in diameter supramolecular cup-shaped lipoprotein basket at the cell PM, where membrane-bound secretory vesicles dock and fuse to release vesicular contents.