Hyaluronan Export through Plasma Membranes Depends on Concurrent K+ Efflux by Kir Channels

Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K+ channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl2 which all belong to ATP-sensitive inwardly-rectifying Kir channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K+ channels Kir3.4 and Kir6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K+ efflux

The Use of Anti-VDAC2 Antibody for the Combined Assessment of Human Sperm Acrosome Integrity and Ionophore A23187-Induced Acrosome Reaction

Voltage-dependent anion channel (VDAC) is mainly located in the mitochondrial outer membrane and participates in many biological processes. In mammals, three VDAC subtypes (VDAC1, 2 and 3) have been identified. Although VDAC has been extensively studied in various tissues and cells, there is little knowledge about the distribution and function of VDAC in male mammalian reproductive system. Several studies have demonstrated that VDAC exists in mammalian spermatozoa and is implicated in spermatogenesis, sperm maturation, motility and fertilization. However, there is no knowledge about the respective localization and function of three VDAC subtypes in human spermatozoa. In this study, we focused on the presence of VDAC2 in human spermatozoa and its possible role in the acrosomal integrity and acrosome reaction using specific anti-VDAC2 monoclonal antibody for the first time. The results exhibited that native VDAC2 existed in the membrane components of human spermatozoa. The co-incubation of spermatozoa with anti-VDAC2 antibody did not affect the acrosomal integrity and acrosome reaction, but inhibited ionophore A23187-induced intracellular Ca2+ increase. Our study suggested that VDAC2 was located in the acrosomal membrane or plasma membrane of human spermatozoa, and played putative roles in sperm functions through mediating Ca2+ transmembrane transport

Ca2+ Extrusion by NCX Is Compromised in Olfactory Sensory Neurons of OMP−/− Mice

The role of olfactory marker protein (OMP), a hallmark of mature olfactory sensory neurons (OSNs), has been poorly understood since its discovery. The electrophysiological and behavioral phenotypes of OMP knockout mice indicated that OMP influences olfactory signal transduction. However, the mechanism by which this occurs remained unknown.We used intact olfactory epithelium obtained from WT and OMP(-/-) mice to monitor the Ca(2+) dynamics induced by the activation of cyclic nucleotide-gated channels, voltage-operated Ca(2+) channels, or Ca(2+) stores in single dendritic knobs of OSNs. Our data suggested that OMP could act to modulate the Ca(2+)-homeostasis in these neurons by influencing the activity of the plasma membrane Na(+)/Ca(2+)-exchanger (NCX). Immunohistochemistry verifies colocalization of NCX1 and OMP in the cilia and knobs of OSNs. To test the role of NCX activity, we compared the kinetics of Ca(2+) elevation by stimulating the reverse mode of NCX in both WT and OMP(-/-) mice. The resulting Ca(2+) responses indicate that OMP facilitates NCX activity and allows rapid Ca(2+) extrusion from OSN knobs. To address the mechanism by which OMP influences NCX activity in OSNs we studied protein-peptide interactions in real-time using surface plasmon resonance technology. We demonstrate the direct interaction of the XIP regulatory-peptide of NCX with calmodulin (CaM).Since CaM also binds to the Bex protein, an interacting protein partner of OMP, these observations strongly suggest that OMP can influence CaM efficacy and thus alters NCX activity by a series of protein-protein interactions

A SLOW OUTWARD CURRENT AND A HYPOOSMOLALITY INDUCED ANION CONDUCTANCE IN EMBRYONIC CHICKEN OSTEOCLASTS

In this paper we report on a hypoosmolality induced current, Iosmo, in embryonic chicken osteoclasts, which could only be studied when blocking a simultaneously active, unidentified slow outward cur- rent, Islo. Islo was observed in all of the examined cells when both the intracellular and extracellular solutions contained sodium as the major cation and no potassium. The current was outwardly rectifying and acti- vated at membrane potentials more positive than +44 ± 12 mV (n = 31). The time to half activation of the current was also voltage dependent and was 350 ms at Vm = +80 mV, and 78 ms at Vm = +120 mV. The current did not inactivate during periods up to 5 s. Extracellular 4-AP (5 mM), TEA (5 mM) and Ba 2+ (1 mM), blockers of K + conductances in chicken osteoclasts, did not influence Islo. However, Islo was inhibited by 50 mM extracellular verapamil, which allowed us to study Iosmo in isolation. Exposure of the osteoclasts to hypotonic solution resulted in the development of a depolarization acti- vated Iosmo. It developed after a 1-min delay and reached its maximum within 10 minutes. Half-maximal activation occurred after 4.4 ± 0.9 min (n = 9). The current activated within a few ms upon depolariza- tion and did not inactivate during at least 5 sec. Iosmo reversed around the calculated Nernst potential for C1 Ś (ECl = +7.3 mV and Vrev = +5.4 ± 3.6 mV, n = 9). The underlying conductance, Gosmo exhibited mod- erate outward rectification around 0 mV in symmetrical C1 Ś solutions. Ion substitution experiments showed that Gosmo is an anion conductance with PCl » PF ≯ Pgluc ≯≯ P Na. Iosmo was blocked by 0.5 mM SITS but 50 mM verapamil, 5 mM TEA, 5 mM 4-AP, 1 mM Ba 2+, 50 mM cytochalasin D and 0.5 mM alendronate did not have any effect on the current. Cl Ś current