The plasma membrane expression of the rat brain calcium channel subunits alpha1A, alpha2-delta and the beta subunits beta1b, beta2a, beta3b and beta4 was examined by transient expression in COS-7 cells. Neither alpha1A nor alpha2-delta localized to the plasma membrane, either alone or when coexpressed. However, coexpression of alpha1A or alpha2-delta/alpha1A with any of the beta subunits caused alpha1A and alpha2 to be targetted to the plasma membrane. The alpha1A antibody is directed against an exofacial epitope at the mouth of the pore, which is not exposed unless cells are depolarized, both for native alpha1A channels in dorsal root ganglion neurons and for alpha1A expressed with a beta subunit. This subsidiary result provides evidence that either channel opening or inactivation causes a conformational change at the mouth of the pore of alpha1A. Immunostaining for alpha1A was obtained in depolarized non-permeabilized cells, indicating correct orientation in the membrane only when it was coexpressed with a beta subunit. In contrast, beta1b and beta2a were associated with the plasma membrane when expressed alone. However, this is not a prerequisite to target alpha1A to the membrane since beta3 and beta4 alone showed no differential localization, but did direct the translocation of alpha1A to the plasma membrane, suggesting a chaperone role for the beta subunits.

These data suggest that mGluRs and GABA(B)Rs play a role in the regulation of the endocrine pancreas with mechanisms probably involving direct activation or inhibition of voltage dependent Ca(2+)-channels, cAMP generation and G-protein-mediated modulation of K(ATP) channels.

We report for the first time abnormalities in cardiac ventricular electrophysiology in a genetically modified murine model lacking the Scn3b gene (Scn3b−/−). Scn3b−/− mice were created by homologous recombination in embryonic stem (ES) cells. RT-PCR analysis confirmed that Scn3b mRNA was expressed in the ventricles of wild-type (WT) hearts but was absent in the Scn3b−/− hearts. These hearts also showed increased expression levels of Scn1b mRNA in both ventricles and Scn5a mRNA in the right ventricles compared to findings in WT hearts. Scn1b and Scn5a mRNA was expressed at higher levels in the left than in the right ventricles of both Scn3b−/− and WT hearts. Bipolar electrogram and monophasic action potential recordings from the ventricles of Langendorff-perfused Scn3b−/− hearts demonstrated significantly shorter ventricular effective refractory periods (VERPs), larger ratios of electrogram duration obtained at the shortest and longest S1–S2 intervals, and ventricular tachycardias (VTs) induced by programmed electrical stimulation. Such arrhythmogenesis took the form of either monomorphic or polymorphic VT. Despite shorter action potential durations (APDs) in both the endocardium and epicardium, Scn3b−/− hearts showed ΔAPD90 values that remained similar to those shown in WT hearts. The whole-cell patch-clamp technique applied to ventricular myocytes isolated from Scn3b−/− hearts demonstrated reduced peak Na+ current densities and inactivation curves that were shifted in the negative direction, relative to those shown in WT myocytes. Together, these findings associate the lack of the Scn3b gene with arrhythmic tendencies in intact perfused hearts and electrophysiological features similar to those in Scn5a+/− hearts.

Amphisomes are organelles of the autophagy pathway that result from the fusion of autophagosomes with late endosomes. While biogenesis of autophagosomes and late endosomes occurs continuously at axon terminals, non-degradative roles of autophagy at boutons are barely described. Here, we show that in neurons BDNF/TrkB traffick in amphisomes that signal locally at presynaptic boutons during retrograde transport to the soma. This is orchestrated by the Rap GTPase-activating (RapGAP) protein SIPA1L2, which connects TrkB amphisomes to a dynein motor. The autophagosomal protein LC3 regulates RapGAP activity of SIPA1L2 and controls retrograde trafficking and local signaling of TrkB. Following induction of presynaptic plasticity, amphisomes dissociate from dynein at boutons enabling local signaling and promoting transmitter release. Accordingly, sipa1l2 knockout mice show impaired BDNF-dependent presynaptic plasticity. Taken together, the data suggest that in hippocampal neurons, TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity.

Voltage-dependent calcium channels (VDCCs) are heteromeric complexes consisting of a channel-forming á1 subunit and accessory á2-ä and â subunits. There are at least eight cloned and expressed á1 subunits (Perez-Reyes & Schneider, 1994;Perez-Reyes et al. 1998), at least six of which (á1A-E and G) are found in the nervous system. The á1 subunit determines the characteristics of the channel and some of the cloned channels have been attributed to functionally identified channels. The N-type channel is believed to be encoded by the á1B clone (De Waard et al. 1994), PÏQ-type channels by á1A (Gillard et al. 1997) and L-type channels by á1C and á1D (Birnbaumer et al. 1994). The assignment of the á1E clone to a native channel has been controversial. It has been suggested that it encodes either an R-type (residual) (Randall & Tsien, 1995) or a subset of low voltageactivated T-type channels (Bourinet et al. 1996). The á1G subunit encodes a T-type channel (Perez-Reyes et al. 1998). The accessory subunits, particularly the intracellular â subunit, have been shown to have marked effects on the properties of á1 subunits (apart from á1G), including modification of kinetics, amplitude and targeting of the complex to the plasma membrane (Singer et al. 1991;. There are four â subunits, all of which are expressed in the nervous system (Perez-Reyes & Schneider, 1994). We have shown previously that the â subunit has a chaperone-like effect, promoting functional expression of the VDCC á1A subunit at the plasma membrane of COS_7 cells (Brice et al. 1997), and similar results have been obtained for cardiac á1C with â2a (Chien et al. 1995). In neurons, and other polarized cells, VDCCs must be sorted to the correct membrane domain for the proper functioning of the cell, as the different subtypes of VDCC have very different biophysical properties and potential for modulation (Dolphin, 1998). For example, in neurons certain subtypes of VDCC are essential to provide Ca¥ for neurotransmitter release. Studies have shown that the N-and PÏQ-type channels are particularly involved, either individually or in combination with each other (Turner et al. 1993;Wheeler et al. 1994), indicating a presynaptic localization for these channels. Nevertheless, these VDCCs have also been

AimIn contrast to extensive reports on the roles of Nav1.5 α-subunits, there have been few studies associating the β-subunits with cardiac arrhythmogenesis. We investigated the sino-atrial and conduction properties in the hearts of Scn3b−/− mice.MethodsThe following properties were compared in the hearts of wild-type (WT) and Scn3b−/− mice: (1) mRNA expression levels of Scn3b, Scn1b and Scn5a in atrial tissue. (2) Expression of the β3 protein in isolated cardiac myocytes. (3) Electrocardiographic recordings in intact anaesthetized preparations. (4) Bipolar electrogram recordings from the atria of spontaneously beating and electrically stimulated Langendorff-perfused hearts.ResultsScn3b mRNA was expressed in the atria of WT but not Scn3b−/− hearts. This was in contrast to similar expression levels of Scn1b and Scn5a mRNA. Immunofluorescence experiments confirmed that the β3 protein was expressed in WT and absent in Scn3b−/− cardiac myocytes. Lead I electrocardiograms from Scn3b−/− mice showed slower heart rates, longer P wave durations and prolonged PR intervals than WT hearts. Spontaneously beating Langendorff-perfused Scn3b−/− hearts demonstrated both abnormal atrial electrophysiological properties and evidence of partial or complete dissociation of atrial and ventricular activity. Atrial burst pacing protocols induced atrial tachycardia and fibrillation in all Scn3b−/− but hardly any WT hearts. Scn3b−/− hearts also demonstrated significantly longer sinus node recovery times than WT hearts.ConclusionThese findings demonstrate, for the first time, that a deficiency in Scn3b results in significant atrial electrophysiological and intracardiac conduction abnormalities, complementing the changes in ventricular electrophysiology reported on an earlier occasion.

SummaryAn in vivo electrophysiological characterisation of the pro- and anti-nociceptive effects of TRPM8 agonists reveals menthol and a novel TRPM8 agonist have selective inhibitory effects on cold sensitivity in neuropathic rats.

The rat brain alpha1A calcium channel clone has been expressed in COS-7 cells together with the neuronal accessory subunits beta1b and alpha2-delta. From reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry and electrophysiology experiments, we have obtained no evidence that these cells contain any endogenous calcium channels. Transfected cells were identified by co-expression of a cDNA for the reporter Green Fluorescent Protein. From immunocytochemical evidence, a high degree of co-expression was obtained between Green Fluorescent Protein and individual calcium channel subunits. When all three calcium channel subunits (alpha1, alpha2-delta and beta1b) were co-expressed, evidence was obtained that all subunits were present at the cell membrane. Voltage-dependent calcium currents were observed between 24 and 72 h after transfection with the three calcium channel subunits. The current density for the combination alpha1A/alpha2-delta/beta1b was 4.19 +/- 0.69 pA.pF(-1) and the current produced was slowly inactivating. The time constant of inactivation of the maximum I(Ba) was 332 +/- 46 ms (n = 5). The voltage-dependence of activation and steady-state inactivation had voltages of half activation and inactivation of 9.5 +/- 2.5 mV and -30.4 +/- 1.5 mV respectively, and there was little overlap between the two curves. The alpha1A current was completely blocked by 100 microM Cd2+ and was also blocked by omega-conotoxin MVIIC (500 nM). Dose-inhibition curves and analysis of k(on) and k(off) for omega-agatoxin IVA both revealed apparent K(D) values of approximately 11 nM for alpha1A currents, with a k(on) of 7.8 x 10(4) M(-1).s(-1). The results suggest that alpha1A expressed in these cells has some resemblance to the P type component of calcium current observed in native neurons, although it shows a somewhat greater degree of inactivation than native P current, more similar to the Q type current component. It also has an affinity for omega-agatoxin IVA 2-5 fold lower than reported for P current, but approximately 9-fold higher than reported for Q current.