The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels. We investigated a prominent voltagedependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in Saccharomyces cerevisiae. Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (

Ca 2؉ is released from the vacuole into the yeast cytoplasm on an osmotic upshock, but how this upshock is perceived was unknown. We found the vacuolar channel, Yvc1p, to be mechanosensitive, showing that the Ca 2؉ conduit is also the sensing molecule. Although fragile, the yeast vacuole allows limited direct mechanical examination. Pressures at tens of millimeters of Hg (1 mmHg ‫؍‬ 133 Pa) activate the 400-pS Yvc1p conductance in whole-vacuole recording mode as well as in the excised cytoplasmic-side-out mode. Raising the bath osmolarity activates this channel and causes vacuolar shrinkage and deformation. It appears that, on upshock, a transient osmotic force activates Yvc1p to release Ca 2؉ from the vacuole. Mechanical activation of Yvc1p occurs regardless of Ca 2؉ concentration and is apparently independent of its known Ca 2؉ activation, which we now propose to be an amplification mechanism (Ca 2؉ -induced Ca 2؉ release). Yvc1p is a member of the transient receptor potential-family channels, several of which have been associated with mechanosensation in animals. The possible use of Yvc1p as a molecular model to study mechanosensation in general is discussed.

Sigma receptors modulate the excitability of peptidergic nerve terminals in the neurohypophysis by inhibiting voltage-dependent K¤ channels (IK) (Wilke et al. 1999a). The activation of sigma receptors by a variety of ligands reduces current flow through two distinct K¤ channel types, the A-current channel (IA) and the Ca¥-activated K¤ channel (IBK). Current is reduced by the same proportion over the entire accessible voltage range, with no shift in the voltage dependence of activation or inactivation. Furthermore, the residual unblocked currents inactivate with very similar rates, indicating that sigma receptor modulation entails a shutting down of function rather than a modification of gating behaviour (Wilke et al. 1998(Wilke et al. , 1999a. A comparison of the concentration dependence of IA reduction with that of IBK reduction indicated that the sigma receptor ligand PPHT inhibits both of these channels with a very similar EC50 (Wilke et al. 1998); similar results were obtained with haloperidol (Wilke et al. 1999a). Both IA and IK were reduced proportionally by a large number of sigma receptor ligands (including ditolylguanidine, SKF10047, pentazocine, haloperidol, PPHT, U101958, and apomorphine), suggesting that in the rat the same receptor is coupled to two types of K¤ channels. In Dµ, D× and DÚ dopamine receptor-deficient mice, sigma receptor ligands reduced IK as effectively as in wild-type mice, indicating that the responses are not mediated by dopamine receptor subtypes known to interact with some sigma receptor ligands (Wilke et al. 1999a 1. Receptor-mediated modulation of ion channels generally involves G-proteins, phosphorylation, or both in combination. The sigma receptor, which modulates voltage-gated K¤ channels, is a novel protein with no homology to other receptors known to modulate ion channels. In the present study patch clamp and photolabelling techniques were used to investigate the mechanism by which sigma receptors modulate K¤ channels in peptidergic nerve terminals. 2. The sigma receptor photoprobe iodoazidococaine labelled a protein with the same molecular mass (26 kDa) as the sigma receptor protein identified by cloning. 3. The sigma receptor ligands pentazocine and SKF10047 modulated K¤ channels, despite intra-terminal perfusion with GTP-free solutions, a G-protein inhibitor (GDPâS), a G_protein activator (GTPãS) or a non-hydrolysable ATP analogue (AMPPcP). 4. Channels in excised outside-out patches were modulated by ligand, indicating that soluble cytoplasmic factors are not required. In contrast, channels within cell-attached patches were not modulated by ligand outside a patch, indicating that receptors and channels must be in close proximity for functional interactions. Channels expressed in oocytes without receptors were unresponsive to sigma receptor agonists, ruling out inhibition through a direct drug interaction with channels. 5. These experiments indicate that sigma receptor-mediated signal transduction is membrane delimited, and requires neither G-protein activation nor prot...

Traumatic brain injury (TBI) results in long-lasting cognitive impairments for which there is currently no accepted treatment. A well-established mouse model of mild to moderate TBI, lateral fluid percussion injury (FPI), shows changes in network excitability in the hippocampus including a decrease in net synaptic efficacy in area CA1 and an increase in net synaptic efficacy in dentate gyrus. Previous studies identified a novel therapy consisting of branched chain amino acids (BCAAs), which restored normal mouse hippocampal responses and ameliorated cognitive impairment following FPI. However, the optimal BCAA dose and length of treatment needed to improve cognitive recovery is unknown. In the current study, mice underwent FPI then consumed 100 mM BCAA supplemented water ad libitum for 2, 3, 4, 5, and 10 days. BCAA therapy ameliorated cognitive impairment at 5 and 10 days duration. Neither BCAA supplementation at 50 mM nor BCAAs when dosed 5 days on then 5 days off was sufficient to ameliorate cognitive impairment. These results suggest that brain injury causes alterations in hippocampal function, which underlie and contribute to hippocampal cognitive impairment, which are reversible with at least 5 days of BCAA treatment, and that sustaining this effect is dependent on continuous treatment. Our findings have profound implications for the clinical investigation of TBI therapy.

The neurological impairments associated with traumatic brain injury include learning and memory deficits and increased risk of seizures. The hippocampus is critically involved in both of these phenomena and highly susceptible to damage by traumatic brain injury. To examine network activity in the hippocampal CA1 region after lateral fluid percussion injury, we used a combination of voltage-sensitive dye, field potential, and patch clamp recording in mouse hippocampal brain slices. When the stratum radiatum (SR) was stimulated in slices from injured mice, we found decreased depolarization in SR and increased hyperpolarization in stratum oriens (SO), together with a decrease in the percentage of pyramidal neurons firing stimulus-evoked action potentials. Increased hyperpolarization in SO persisted when glutamatergic transmission was blocked. However, we found no changes in SO responses when the alveus was stimulated to directly activate SO. These results suggest that the increased SO hyperpolarization evoked by SR stimulation was mediated by interneurons that have cell bodies and/or axons in SR, and form synapses in stratum pyramidale and SO. A low concentration (100 nM) of the synthetic cannabinoid WIN55,212-2, restored CA1 output in slices from injured animals. These findings support the hypothesis that increased GABAergic signaling by cannabinoid-sensitive interneurons contributes to the reduced CA1 output following traumatic brain injury.