Regulation of 1-Type Calcium Channels by the Rem2 GTPase in Pancreatic Bata Cells

Specific Aims Non-insulin dependent diabetes mellitus (NIDDM) or type II diabetes is a major health problem in the US. Type II diabetes is characterized by high glucose levels in the blood. This abnormal state is the result of the failure of two mechanisms that maintain glucose homeostasis. First, tissues that normally transport glucose from the blood in response to glucose become resistant to insulin. Second, pancreatic 13 cells lose their ability to secrete insulin in response to glucose challenge [1]. This grant proposal focuses on this latter aspect of the disease. L-type Ca2+ channels playa pivotal role in the regulation of insulin secretion by pancreatic 13 cells. Recent experiments have demonstrated that ras-related GTPases of the Rad/Rem/Rem2/Gem/Kir (RGK) subfamily of ras-related GTPases down-regulate L-type Ca2+ channel activity [2, 3]. Our preliminary data suggests that Rem2 plays an important role in regulating L-type Ca2+ channels in pancreatic 13 cells. The guiding hypothesis of this grant proposal is that Rem2 is involved in long term negative feedback control of L-type Ca2+ channel activity in pancreatic J3 cells through a mechanism that involves glucose stimulation of Rem2 levels. Dysregulation of this previously unrecognized pathway may therefore play an important role in development of NIDDM. In support of this hypothesis the following observations have been made: 1) a micro array experiment identified Rem2 as a gene that is up-regulated in MIN6 insulinoma cells in response to high glucose, 2) Rem2 mRNA levels were confirmed to be up-regulated in response to 0- but not L- glucose in MIN6 Cells, 3) ectopic expression of Rem2 in MIN6 cells inhibits insulin secretion, 4) Rem2 inhibits L-type channel activity in heterologous expression and endogenous systems, 5) Rem2 binds Cav 13 subunits in vivo. Two specific aims are proposed to evaluate the hypothesis. Specific Aim 1 will determine the mechanism by which Rem2 levels are regulated in 13 cells by glucose and whether up-regulation of Rem2 occurs in diabetic mice. Specific Aim 2 will address the mechanism of Rem2 inhibition of L-type Ca2+ channels. Together, these aims will provide new insight into the regulation of L-type calcium channels in pancreatic 13 cells and the pathogenesis of type II diabetes. Specific Aim 1. How does glucose regulate Rem2 levels and is Rem2 up-regulated under diabetic conditions? Glucose treatment of pancreatic 13 cells elicits the activation of a number of signal transduction pathways. Anyone of these could regulate Rem2 levels. The first part of this aim will dissect which one of these pathways is utilized to up-regulate Rem2 levels. The second part of the aim will examine whether Rem2 levels are up-regulated by hyperglycemic conditions in diabetic mice. It is proposed that up regulation of Rem2 by this condition may playa role in the lack of response to glucose by 13 cells in NIDDM. Specific Aim 2. Does Rem2 affect Cay a localization or function at the cell surface and, what is the mechanism of inhibition? Cay 13 subunits bind the Cava subunit to affect channel activity in two distinct manners. First, Cay 13 subunits are required for transporting the pore-forming and voltage sensing Cava subunit of the channel from the ER to the plasma membrane [4]. Second, Cav 13 subunits are thought to regulate channel function at the surface [5-7]. RGK GTPases all can inhibit channel activity by binding Cay 13 subunits [2, 3]. It is therefore possible that Rem2 could affect trafficking of the Cava subunit, inhibit Cava activity at the plasma membrane, or a combination of both. The first part of this aim will address whether Rem2 disrupts the Cava- Cay 13 interaction. The second part of this aim will address whether Rem2 affects surface localization of the Cava subunit.