Differential effects of RGK proteins on L-type channel function in adult mouse skeletal muscle.

Work in heterologous systems has revealed that members of the Rad, Rem, Rem2, Gem/Kir (RGK) family of small GTP-binding proteins profoundly inhibit L-type Ca(2+) channels via three mechanisms: 1), reduction of membrane expression; 2), immobilization of the voltage-sensors; and 3), reduction of Po without impaired voltage-sensor movement. However, the question of which mode is the critical one for inhibition of L-type channels in their native environments persists. To address this conundrum in skeletal muscle, we overexpressed Rad and Rem in flexor digitorum brevis (FDB) fibers via in vivo electroporation and examined the abilities of these two RGK isoforms to modulate the L-type Ca(2+) channel (CaV1.1). We found that Rad and Rem both potently inhibit L-type current in FDB fibers. However, intramembrane charge movement was only reduced in fibers transfected with Rad; charge movement for Rem-expressing fibers was virtually identical to charge movement observed in naïve fibers. This result indicated that Rem supports inhibition solely through a mechanism that allows for translocation of CaV1.1's voltage-sensors, whereas Rad utilizes at least one mode that limits voltage-sensor movement. Because Rad and Rem differ significantly only in their amino-termini, we constructed Rad-Rem chimeras to probe the structural basis for the distinct specificities of Rad- and Rem-mediated inhibition. Using this approach, a chimera composed of the amino-terminus of Rem and the core/carboxyl-terminus of Rad inhibited L-type current without reducing charge movement. Conversely, a chimera having the amino-terminus of Rad fused to the core/carboxyl-terminus of Rem inhibited L-type current with a concurrent reduction in charge movement. Thus, we have identified the amino-termini of Rad and Rem as the structural elements dictating the specific modes of inhibition of CaV1.1.

Potent inhibition of L-type Ca2+ currents by a Rad variant associated with congestive heart failure.

Ca(2+) influx via L-type voltage-gated Ca(2+) channels supports the plateau phase of ventricular action potentials and is the trigger for excitation-contraction (EC) coupling in the myocardium. Rad, a member of the RGK (Rem, Rem2, Rad, Gem/Kir) family of monomeric G proteins, regulates ventricular action potential duration and EC coupling gain through its ability to inhibit cardiac L-type channel activity. In this study, we have investigated the potential dysfunction of a naturally occurring Rad variant (Q66P) that has been associated with congestive heart failure in humans. Specifically, we have tested whether Rad Q66P limits, or even eliminates, the inhibitory actions of Rad on CaV1.2 and CaV1.3, the two L-type channel isoforms known to be expressed in the heart. We have found that mouse Rad Q65P (the murine equivalent of human Rad Q66P) inhibits L-type currents conducted by CaV1.2 or CaV1.3 channels as potently as wild-type Rad (>95% inhibition of both channels). In addition, Rad Q65P attenuates the gating movement of both channels as effectively as wild-type Rad, indicating that the Q65P substitution does not differentially impair any of the three described modes of L-type channel inhibition by RGK proteins. Thus, we conclude that if Rad Q66P contributes to cardiomyopathy, it does so via a mechanism that is not related to its ability to inhibit L-type channel-dependent processes per se. However, our results do not rule out the possibility that decreased expression, mistargeting or altered regulation of Rad Q66P may reduce the RGK protein's efficacy in vivo.

A functional role for adult hippocampal neurogenesis in spatial pattern separation.

The dentate gyrus (DG) of the mammalian hippocampus is hypothesized to mediate pattern separation-the formation of distinct and orthogonal representations of mnemonic information-and also undergoes neurogenesis throughout life. How neurogenesis contributes to hippocampal function is largely unknown. Using adult mice in which hippocampal neurogenesis was ablated, we found specific impairments in spatial discrimination with two behavioral assays: (i) a spatial navigation radial arm maze task and (ii) a spatial, but non-navigable, task in the mouse touch screen. Mice with ablated neurogenesis were impaired when stimuli were presented with little spatial separation, but not when stimuli were more widely separated in space. Thus, newborn neurons may be necessary for normal pattern separation function in the DG of adult mice.

Age-dependent and -independent behavioral deficits in Tg2576 mice.

The Tg2576 mouse model of excessive cerebral beta-amyloid deposition is now more than a decade old, yet consensus as to its exact characteristics and utility as a model of Alzheimer's disease is still lacking. Four different cohorts of control and Tg2576 mice, aged approximately 3, 9, 13 and 21 months, were therefore subjected to a battery of tests, principally to assess cognitive and species-typical behaviors. A novel test, the paddling Y-maze, demonstrated an age-dependent deficit in 10 and 14, but not 3 month Tg2576 mice, also in aged (21 month) control mice. However, in many other cognitive tests few Tg2576-related deficits could be shown. This frequently seemed attributable to poor performance of control mice. Tests of species-typical behaviors showed that Tg2576 mice had a deficit in burrowing behavior at all ages. An age-independent deficit was also seen in nest construction, but only when mice were group-housed; most individually housed mice in either group made reasonable nests. Overall, the results suggested that these Tg2576 mice are not a simple, suitable or reliable model for routine screening of treatments for Alzheimer's disease. However, this model might perform better behaviorally on a different genetic background.