Background— Impairment of intracellular Ca 2+ homeostasis and mitochondrial function has been implicated in the development of cardiomyopathy. Mitochondrial Ca 2+ uptake is thought to be mediated by the Ca 2+ uniporter (MCU) and a thus far speculative non-MCU pathway. However, the identity and properties of these pathways are a matter of intense debate, and possible functional alterations in diseased states have remained elusive. Methods and Results— By patch clamping the inner membrane of mitochondria from nonfailing and failing human hearts, we have identified 2 previously unknown Ca 2+ -selective channels, referred to as mCa1 and mCa2. Both channels are voltage dependent but differ significantly in gating parameters. Compared with mCa2 channels, mCa1 channels exhibit a higher single-channel amplitude, shorter openings, a lower open probability, and 3 to 5 subconductance states. Similar to the MCU, mCa1 is inhibited by 200 nmol/L ruthenium 360, whereas mCa2 is insensitive to 200 nmol/L ruthenium 360 and reduced only by very high concentrations (10 μmol/L). Both mitochondrial Ca 2+ channels are unaffected by blockers of other possibly Ca 2+ -conducting mitochondrial pores but were activated by spermine (1 mmol/L). Notably, activity of mCa1 and mCa2 channels is decreased in failing compared with nonfailing heart conditions, making them less effective for Ca 2+ uptake and likely Ca 2+ -induced metabolism. Conclusions— Thus, we conclude that the human mitochondrial Ca 2+ uptake is mediated by these 2 distinct Ca 2+ channels, which are functionally impaired in heart failure. Current properties reveal that the mCa1 channel underlies the human MCU and that the mCa2 channel is responsible for the ruthenium red–insensitive/low-sensitivity non-MCU–type mitochondrial Ca 2+ uptake.

Current therapy for inflammatory pain includes the peripheral application of opioid receptor agonists. Activation of opioid receptors modulates voltage-gated ion channels, but it is unclear whether opioids can also influence ligand-gated ion channels [e.g., the transient receptor potential vanilloid type 1 (TRPV1)]. TRPV1 channels are involved in the development of thermal hypersensitivity associated with tissue inflammation. In this study, we investigated -opioid receptor and TRPV1 expression in primary afferent neurons in the dorsal root ganglion (DRG) in complete Freund's adjuvant (CFA)-induced paw inflammation. In addition, the present study examined whether the activity of TRPV1 in DRG neurons can be inhibited by -opioid receptor (-receptor) ligands and whether this inhibition is increased after CFA inflammation. Immunohistochemistry demonstrated colocalization of TRPV1 and -receptors in DRG neurons. CFA-induced inflammation increased significantly the number of TRPV1-and -receptor-positive DRG neurons, as well as TRPV1 binding sites. In whole-cell patch clamp studies, opioids significantly decreased capsaicin-induced TRPV1 currents in a naloxone-and pertussis toxinsensitive manner. The inhibitory effect of morphine on TRPV1 was abolished by forskolin and 8-bromo-cAMP. During inflammation, an increase in TRPV1 is apparently rivaled by an increase of -receptors. However, in single dissociated DRG neurons, the inhibitory effects of morphine are not different between animals with and without CFA inflammation. In in vivo experiments, we found that locally applied morphine reduced capsaicin-induced thermal allodynia. In summary, our results indicate that -receptor activation can inhibit the activity of TRPV1 via G i/o proteins and the cAMP pathway. These observations demonstrate an important new mechanism underlying the analgesic efficacy of peripherally acting -receptor ligands in inflammatory pain.

UC. Effects of KCNE2 on HCN isoforms: distinct modulation of membrane expression and single channel properties. Am J Physiol Heart Circ Physiol 297: H355-H363, 2009. First published May 8, 2009 doi:10.1152/ajpheart.00154.2009.-Hyperpolarization-activated cation (HCN) channels give rise to an inward current with similar but not identical characteristics compared with the pacemaker current (I f), suggesting that HCN channel function is modulated by regulatory ␤-subunits in native tissue. KCNE2 has been proposed to serve as a ␤-subunit of HCN channels; however, available data remain contradictory. To further clarify this situation, we therefore analyzed the effect of KCNE2 on whole cell currents, single channel properties, and membrane protein expression of all cardiac HCN isoforms in the CHO cell system. On the whole cell level, current densities of all HCN isoforms were significantly increased by KCNE2 without altering voltage dependence or current reversal. While these results correlated well with the KCNE2-mediated 2.2-fold and 1.6-fold increases of membrane protein levels of HCN2 and HCN4, respectively, no effect of KCNE2 on HCN1 expression was obtained. All HCN subtypes displayed faster activation kinetics upon coexpression with KCNE2. Most importantly, for the first time, we demonstrated modulation of single channel function by KCNE2, thus supporting direct functional interaction with HCN subunits. In the presence of KCNE2, the single channel amplitudes and conductance of HCN1, HCN2, and HCN4 were significantly increased versus control recordings. Mean open time was significantly increased in cells coexpressing HCN2 ϩ KCNE2, whereas it was unaffected in HCN1 ϩ KCNE2 cotransfected cells and reduced in HCN4 ϩ KCNE2 cotransfected cells compared with the respective HCN subunits alone. Thus, we demonstrate KCNE2-mediated distinct effects on HCN membrane expression and direct functional modulation of HCN isoforms, further supporting that KCNE2 surves as a regulatory ␤-subunit of HCN channels. pacemaker current; hyperpolarization-activated cation; MinK-related peptide; electrophysiology THE HYPERPOLARIZATION-ACTIVATED CATION (HCN) channel current (I f ; also known as I h ) has been identified in various regions of the mammalian heart, brain, peripheral nervous system, and eye (3). In the sinus node, Purkinje cells, and neonatal cardiomyocytes, I f is considered to contribute significantly to spontaneous pacemaker activity (7,8,28). An I f -like current has also been recorded in ventricular and atrial myocytes of mammalian species and in human myocardial tissue (10,11,31).Four mammalian HCN isoforms (HCN1-HCN4) that encode the channels underlying I f have been cloned, three of which are present in the heart (HCN1, HCN2, and HCN4) (16,17,26). The expression of these isoforms varies among species, cardiac regions, and developmental stages (24, 27). In heterologous expression, all HCN channels give rise to a hyperpolarization-activated inward current with similar but not identical characteristics compared with native ...

Hyperalgesia is a cardinal symptom of opioid withdrawal. The transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated ion channel expressed on sensory neurons responding to noxious heat, protons, and chemical stimuli such as capsaicin. TRPV1 can be inhibited via μ-opioid receptor (MOR)-mediated reduced activity of adenylyl cyclases (ACs) and decreased cyclic adenosine monophosphate (cAMP) levels. In contrast, opioid withdrawal following chronic activation of MOR uncovers AC superactivation and subsequent increases in cAMP and protein kinase A (PKA) activity. Here we investigated (1) whether an increase in cAMP during opioid withdrawal increases the activity of TRPV1 and (2) how opioid withdrawal modulates capsaicin-induced nocifensive behavior in rats. We applied whole-cell patch clamp, microfluorimetry, cAMP assays, radioligand binding, site-directed mutagenesis, and behavioral experiments. Opioid withdrawal significantly increased cAMP levels and capsaicin-induced TRPV1 activity in both transfected human embryonic kidney 293 cells and dissociated dorsal root ganglion (DRG) neurons. Inhibition of AC and PKA, as well as mutations of the PKA phosphorylation sites threonine 144 and serine 774, prevented the enhanced TRPV1 activity. Finally, capsaicin-induced nocifensive behavior was increased during opioid withdrawal in vivo. In summary, our results demonstrate an increased activity of TRPV1 in DRG neurons as a new mechanism contributing to opioid withdrawal-induced hyperalgesia.

In the Results section, the description of the data for Figure 4G is incorrect. The correct sentence is below.Jnk2 +/+ mice showed a stronger signal than Jnk2 -/-mice ( Figure 4G), which indicates that TNF induced more intestinal permeability in the Jnk2 +/+ mouse.The authors regret the error.