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Connexin46 (Cx46), together with Cx50, forms gap junction channels between lens fibers and participates in the lens pump-leak system, which is essential for the homeostasis of this avascular organ. Mutations in Cx50 and Cx46 correlate with cataracts, but the functional relationship between the mutations and cataract formation is not always clear. Recently, it was found that a mutation at the third position of hCx46 that substituted an aspartic acid residue with a tyrosine residue (hCx46D3Y) caused an autosomal dominant zonular pulverulent cataract. We expressed EGFP-labeled hCx46wt and hCx46D3Y in HeLa cells and found that the mutation did not affect the formation of gap junction plaques. Dye transfer experiments using Lucifer Yellow (LY) and ethidium bromide (EthBr) showed an increased degree of dye coupling between the cell pairs expressing hCx46D3Y in comparison to the cell pairs expressing hCx46wt. In Xenopus oocytes, two-electrode voltage-clamp experiments revealed that hCx46wt formed voltage-sensitive hemichannels. This was not observed in the oocytes expressing hCx46D3Y. The replacement of the aspartic acid residue at the third position by another negatively charged residue, glutamic acid, to generate the mutant hCx46D3E, restored the voltage sensitivity of the resultant hemichannels. Moreover, HeLa cell pairs expressing hCx46D3E and hCx46wt showed a similar degree of dye coupling. These results indicate that the negatively charged aspartic acid residue at the third position of the N-terminus of hCx46 could be involved in the determination of the degree of metabolite cell-to-cell coupling and is essential for the voltage sensitivity of the hCx46 hemichannels.

The C-terminus (CT) of rCx46 consists of 186 residues (H230-I416). Recent studies showed that rCx46(28.2), truncated after H243, altered the formation of functional hemichannels when expressed in Xenopus oocytes, while rCx46(37.7), truncated after A333 formed gap junction hemichannels similarly to rCx46(wt). To analyze the role of the CT up to A333 in functional expression with cell imaging and dye-transfer techniques, different mutants were generated by C-terminal truncation between H243-A333, labeled with EGFP and expressed in HeLa cells. These rCx46 variants were characterized according to their compartmentalization in organelles, their presence in microscopic detectable vesicles and their ability to form gap junction plaques. rCx46 truncated after A311 (rCx46(35.3)) was compartmentalized, was found in vesicles and formed functional gap junction plaques similarly to rCx46(wt). With a truncation after P284 (rCx46(32.6)), the protein was not compartmentalized and the amount of vesicles containing the protein were reduced; however, functional gap junction plaque formation was not affected as compared to rCx46(35.3). rCx46(28.2) did not form functional gap junction plaques; it was not found in vesicles or in cellular compartments. Live-cell imaging and detection of annular junctions for rCx46(32.6) and rCx46(35.3) revealed that the truncation after P284 reduced the frequency of vesicle budding from gap junction plaques and the formation of annular junctions. These results suggest that the C-terminal region of rCx46 up to A311 (rCx46(35.3)) is necessary for its correct compartmentalization and internalization in the form of annular junctions, while the H230-P284 C-terminal region (rCx46(32.6)) is sufficient for the formation of dye coupled gap junction channels.

Whole-cell patch-clamp analysis revealed a resting membrane potential of −60 mV in primary osteoblasts and in the MG-63 osteoblast-like cells. Depolarization-induced action potentials were characterized by duration of 60 ms, a minimal peak-to-peak distance of 180 ms, a threshold value of −20 mV and a repolarization between the spikes to −45 mV. Expressed channels were characterized by application of voltage pulses between −150 mV and 90 mV in 10 mV steps, from a holding potential of −40 mV. Voltages below −60 mV induced an inward current. Depolarizing voltages above −30 mV evoked two currents: (a) a fast activated and inactivated inward current at voltages between −30 and 30 mV, and (b) a delayed-activated outward current that was induced by voltages above −30 mV. Electrophysiological and pharmacological parameters indicated that hyperpolarization activated strongly rectifying K+ (Kir) channels, whereas depolarization activated tetrodotoxin sensitive voltage gated Na+ (Nav) channels as well as delayed, slowly activated, non-inactivating, and tetraethylammonium sensitive voltage gated K+ (Kv) channels. In addition, RT-PCR showed expression of Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, and Kir2.1, Kir2.3, and Kir2.4 as well as Kv2.1. We conclude that osteoblasts express channels that allow firing of action potentials. The final publication is available at Springer via https://doi.org/10.1007/s10863-011-9354-7. BMBF/NANOTOME DFG/Transregio37 Boehringer Ingelheim International GmbH BMBF/NANOTOME DFG/Transregio/37 Boehringer Ingelheim International GmbH

Purinergic signalling in rat GFSHR-17 granulosa cells was characterised by Ca(2+)-imaging and perforated patch-clamp. We observed a resting intracellular Ca(2+)-concentration ([Ca(2+)](i)) of 100 nM and a membrane potential of -40 mV. This was consistent with high K(+)- and Cl(-) permeability and a high intracellular Cl(-) concentration of 40 mM. Application of ATP for 5-15 s every 3 min induced repeated [Ca(2+)](i) increases and a 30 mV hyperpolarization. The phospholipase C inhibitor U73122 or the IP(3)-receptor antagonist 2-aminoethoethyl diphenyl borate suppressed ATP responses. Further biochemical and pharmacological experiments revealed that ATP responses were related to stimulation of P2Y(2) and P2Y(4) receptors and that the [Ca(2+)](i) increase was a prerequisite for hyperpolarization. Inhibitors of Ca(2+)-activated channels or K(+) channels did not affect the ATP-evoked responses. Conversely, inhibitors of Cl(-) channels hyperpolarized cells to -70 mV and suppressed further ATP-evoked hyperpolarization. We propose that P2Y(2) and P2Y(4) receptors in granulosa cells modulate Cl(-) permeability by regulating Ca(2+)-release.