Faculty Bibliography

Polycystin-2 (PC2), a member of the transient receptor potential family of ion channels (TRPP2), forms a calcium-permeable cation channel. Mutations in PC2 lead to polycystic kidney disease. From the primary sequence and by analogy with other channels in this family, PC2 is modeled to have six transmembrane domains. However, most of the structural features of PC2, such as how large the channel is and how many subunits make up the pore of the channel, are unknown. In this study, we estimated the pore size of PC2 from the permeation properties of the channel. Organic cations of increasing size were used as current carriers through the PC2 channel after PC2 was incorporated into lipid bilayers. We found that dimethylamine, triethylamine, tetraethylammonium, tetrabutylammonium, tetrapropylammonium, and tetrapentylammonium were permeable through the PC2 channel. The slope conductance of the PC2 channel decreased as the ionic diameter of the organic cation increased. For each organic cation tested, the currents were inhibited by gadolinium and anti-PC2 antibody. Using the dimensions of the largest permeant cation, the minimum pore diameter of the PC2 channel was estimated to be at least 11 A. The large pore size suggests that the primary state of this channel found in vivo is closed to avoid rundown of cation gradients across the plasma membrane and excessive calcium leak from endoplasmic reticulum stores.

Polycystin-2 (PC-2) is a non-selective cation channel that, when mutated, results in autosomal dominant polycystic kidney disease. In an effort to understand the regulation of this channel, we investigated the role of protein phosphorylation in PC-2 function. We demonstrated the direct incorporation of phosphate into PC-2 in cells and tissues and found that this constitutive phosphorylation occurs at Ser(812), a putative casein kinase II (CK2) substrate domain. Ser(812) can be phosphorylated by CK2 in vitro and substitution S812A results in failure to incorporate phosphate in cultured epithelial cells. Non-phosphorylated forms of PC-2 traffic normally in the endoplasmic reticulum and cilial compartments and retain homo- and hetero-multimerization interactions with PC-2 and polycystin-1, respectively. Single-channel studies of PC-2, S812A, and a substitution mutant, T721A, not related to phosphorylation show that PC-2 and S812A function as divalent cation channels with similar current amplitudes across a range of holding potentials; the T721A channel is not functional. Channel open probabilities for PC-2 and S812A show a bell-shaped dependence on cytoplasmic Ca(2+) but there is a shift in this Ca(2+) dependence such that S812A is 10-fold less sensitive to Ca(2+) activation/inactivation than the wild type PC-2 channel. In vivo analysis of PC-2-dependent enhanced intracellular Ca(2+) transients found that S812A resulted in enhanced transient duration and relative amplitude intermediate between control cells and those overexpressing wild type PC-2. Phosphorylation at Ser(812) modulates PC-2 channel activity and factors regulating this phosphorylation are likely to play a role in the pathogenesis of polycystic kidney disease.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] plays an important physiological role as a precursor for the InsP3-mediated intracellular calcium (Ca2+) signalling cascade. It also regulates membrane trafficking, actin function and transmembrane proteins. SJ-1 (synaptojanin-1), a phosphoinositide phosphatase, regulates the turnover of a PtdIns(4,5)P2 pool involved in clathrin and actin dynamics at the cell surface. We tested the interrelationship of this pool with PtdIns(4,5)P2 pools involved in Ca2+ signalling by expressing in Chinese-hamster ovary cells full-length SJ-1 or its 5-Pase (inositol 5-phosphatase) domain. SJ-1 significantly attenuated the generation of Ca2+ oscillations induced by ATP and the 5-Pase domain mimicked this effect. These changes correlated with increased PtdIns(4,5)P2 phosphatase activity of cellular extracts. Overexpression of the endoplasmic reticulum-anchored PtdIns(4)P phosphatase Sac1 did not affect Ca2+ oscillations, although it increased the Ca2+ efflux rate from intracellular stores. The ability of SJ-1 to alter intracellular Ca2+ signalling indicates a close functional interrelationship between plasma membrane PtdIns(4,5)P2 pools that control actin and endocytosis and those involved in the regulation of specific spatio-temporal Ca2+ signals.

Chromogranins A and B are high capacity, low affinity calcium (Ca(2+)) storage proteins that bind to the inositol 1,4,5-trisphosphate-gated receptor (InsP(3) R). Although most commonly associated with secretory granules of neuroendocrine cells, chromogranins have also been found in the lumen of the endoplasmic reticulum (ER) of many cell types. To investigate the functional consequences of the interaction between the InsP(3) R and the chromogranins, we disrupted the interaction between the two proteins by adding a chromogranin fragment, which competed with chromogranin for its binding site on the InsP(3)R. Responses were monitored at the single channel level and in intact cells. When using InsP(3) R type I incorporated into planar lipid bilayers and activated by cytoplasmic InsP(3) and luminal chromogranin, the addition of the fragment reversed the enhancing effect of chromogranin. Moreover, the expression of the fragment in the ER of neuronally differentiated PC12 cells attenuated agonist-induced intracellular Ca(2+) signaling. These results show that the InsP(3)R/chromogranin interaction amplifies Ca(2+) release from the ER and that chromogranin is an essential component of this intracellular channel complex.

Polycystic kidney disease (PKD) is caused by mutations in two genes, PKD1 and PKD2, which encode for the proteins, polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Although disease-associated mutations have been identified in these two proteins, the sequence of molecular events leading up to clinical symptoms is still unknown. PC1 resides in the plasma membrane and it is thought to function in cell-cell and cell-matrix interactions, whereas PC2 is a calcium (Ca2+) permeable cation channel concentrated in the endoplasmic reticulum. Both proteins localize to the primary cilia where they function as a mechanosensitive receptor complex allowing the entry of Ca2+ into the cell. The downstream signaling pathway involves activation of intracellular Ca2+ release channels, especially the ryanodine receptor (RyR), but subsequent steps are still to be identified. Elucidation of the signaling pathway involved in normal PC1/PC2 function, the functional consequences of PC1/PC2 mutation, and the role of Ca2+ signaling will all help to unravel the molecular mechanisms of cystogenesis in PKD.

Huntingtin, a protein altered by polyglutamine expansion in Huntington's disease (Httexp), forms a signaling complex with the InsP3R, an intracellular calcium channel, and Htt-associated protein 1A (HAP1A). The addition of Httexp increases the InsP3R sensitivity to InsP3, which subsequently makes neurons hyperresponsive to stimulation and presumably more prone to neurodegenerative processes.

The homotetrameric structure of the ryanodine-sensitive intracellular calcium (Ca2+) release channel (ryanodine receptor (RyR)) suggests that the four RyR subunits either combine to form a single pore or that each RyR subunit is an independently conducting pathway. Previously we showed that methanethiosulfonate ethylammonium (MTSEA+) covalently modifies the RyR to reduce current amplitudes in a time-dependent and stepwise manner. To ascertain the number of functionally conducting pores in the RyR, two approaches were combined: modification of the receptor by MTSEA+ and the use of different sized current carriers. Previous reports (Tinker, A., and Williams, A. J. (1993) J. Gen. Physiol. 102, 1107-1129) have shown that the organic cations methylamine, dimethylamine, ethylamine, and trimethylamine are permeant through the RyR but with reduced current amplitude depending upon the diameter of the respective amine. Experiments using the thiol reagent MTSEA+ to modify the channel protein showed that the current amplitudes decrease in steps leading to complete block of the channel when cesium (Cs+) is the current carrier. MTSEA+ modification decreased the number of channel substates as the diameter of the current carrier increased. Comparison of the degree of inhibition of MTSEA+-modified currents allows for differentiation between the two models for channel architecture. These results demonstrate that the conduction pathway for the RyR is comprised of a single central pore.

Chromogranins A and B (CGA and CGB) are high capacity, low affinity calcium (Ca2+) storage proteins found in many cell types most often associated with secretory granules of secretory cells but also with the endoplasmic reticulum (ER) lumen of these cells. Both CGA and CGB associate with inositol 1,4,5-trisphosphate receptor (InsP3R) in a pH-dependent manner. At an intraluminal pH of 5.5, as found in secretory vesicles, both CGA and CGB bind to the InsP3R. When the intraluminal pH is 7.5, as found in the ER, CGA totally dissociates from InsP3R, whereas CGB only partially dissociates. To investigate the functional consequences of the interaction between the InsP3R and CGB monomers or CGA/CGB heteromers, purified mouse InsP3R type I were fused to planar lipid bilayers and activated by 2 microM InsP3. In the presence of luminal CGB monomers or CGA/CGB heteromers the InsP3R/Ca2+ channel open probability and mean open time increased significantly. The channel activity remained elevated when the pH was changed to 7.5, a reflection of CGB binding to the InsP3R even at pH 7.5. These results suggest that CGB may play an important modulatory role in the control of Ca2+ release from the ER. Furthermore, the difference in the ability of CGA and CGB to regulate the InsP3R/Ca2+ channel and the variability of CGA/CGB ratios could influence the pattern of InsP3-mediated Ca2+ release.

Secretory granules of neuroendocrine cells are inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) stores in which the Ca(2+) storage protein, chromogranin A (CGA), couples with InsP(3)-gated Ca(2+) channels (InsP(3)R) located in the granule membrane. The functional aspect of this coupling has been investigated via release studies and planar lipid bilayer experiments in the presence and absence of CGA. CGA drastically increased the release activity of the InsP(3)R by increasing the channel open probability by 9-fold and the mean open time by 12-fold. Our results show that CGA-coupled InsP(3)Rs are more sensitive to activation than uncoupled receptors. This modulation of InsP(3)R channel activity by CGA appears to be an essential component in the control of intracellular Ca(2+) concentration by secretory granules and may regulate the rate of vesicle fusion and exocytosis.

Polycystin-2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease (ADPKD), is the prototypical member of a subfamily of the transient receptor potential (TRP) channel superfamily, which is expressed abundantly in the endoplasmic reticulum (ER) membrane. Here, we show by single channel studies that polycystin-2 behaves as a calcium-activated, high conductance ER channel that is permeable to divalent cations. Epithelial cells overexpressing polycystin-2 show markedly augmented intracellular calcium release signals that are lost after carboxy-terminal truncation or by the introduction of a disease-causing missense mutation. These data suggest that polycystin-2 functions as a calcium-activated intracellular calcium release channel in vivo and that polycystic kidney disease results from the loss of a regulated intracellular calcium release signalling mechanism.