Various human skeletal disorders are thought to be caused by mutations in fibroblast growth factor receptor 3 (FGFR3). These result in chronic FGFR3 hyperactivation and inhibition of bone growth. One such disorder, thanatophoric dysplasia, the most common form of sporadic, lethal dwarfism, is associated frequently with cysteine substitutions (G370C, S371C, and Y373C) in the extracellular juxtamembrane region of the receptor.

Although bisphosphonates have been shown to be potent inhibitors of osteoclast-mediated bone resorption in vivo and in vitro and are used as therapeutic agents in hyper-resorptive bone diseases such as Paget disease or hypercalcemia of malignancy, their exact biochemical target(s) and mode(s) of action are for the most part still unknown. The resorption of bone requires solubilization of the mineral component of the matrix, achieved by acidification of the resorbing compartment by a vacuolar-type proton ATPase (V-ATPase) present in the ruffled border membrane of osteoclasts. Since we have shown that the V-ATPase is inhibited by both ADP and phosphate, which share structural characteristics with bisphosphonates, we hypothesized that inhibition of the osteoclast V-ATPase could be one of the mechanism(s) by which bisphosphonates inhibit bone resorption. Pyrophosphate and the bisphosphonates etidronate, alendronate, and YM-175 inhibited proton transport in membrane vesicles derived from chicken kidney and osteoclasts but with very low potency (IC50 > or = 5 mM). In contrast, the ability of tiludronate to inhibit proton transport was 5-fold higher in kidney-derived vesicles (IC50 = 1.1 mM) and 10,000-fold higher in vesicles derived from osteoclasts (IC50 = 466 nM). Tiludronate also potently inhibited proton transport in yeast microsomal preparations (IC50 = 3.5 microM) and inhibited the activity of purified yeast V-ATPase. The inhibition of the osteoclast V-ATPase-mediated proton transport by tiludronate was rapid, pH-dependent, and reversible. No change in membrane vesicle permeability to protons was detected. The inhibition was noncompetitive with respect to ATP, and tiludronate did not protect the pump from inactivation by N-ethylmaleimide, strongly suggesting that tiludronate does not bind to the catalytic site of the enzyme. It is concluded that tiludronate is a significantly more potent inhibitor of V-ATPase than other bisphosphonates and that it has a significant degree of selectivity for the avian osteoclast V-ATPase relative to the avian kidney V-ATPase.

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The ability of monkeys and rats to carry out spatial working memory tasks has been shown to depend on the persistent firing of pyramidal cells in the prefrontal cortex (PFC), arising from recurrent excitatory connections on dendritic spines. These spines express hyperpolarization-activated cyclic nucleotide-gated (HCN) channels whose open state is increased by cAMP signaling, and which markedly alter PFC network connectivity and neuronal firing. In traditional neural circuits, activation of these non-selective cation channels leads to neuronal depolarization and increased firing rate. Paradoxically, cAMP activation of HCN channels in PFC pyramidal cells reduces working memory-related neuronal firing. This suggests that activation of HCN channels may hyperpolarize rather than depolarize these neurons. The current study tested the hypothesis that Na+influx through HCN channels activates Na+-activated K+(KNaor Slack) channels to hyperpolarize the membrane. We have found that HCN and Slack KNachannels co-immunoprecipitate in cortical extracts and that, by immunoelectron microscopy, they colocalize at postsynaptic spines of PFC pyramidal neurons. A specific blocker of HCN channels, ZD7288, reduces KNacurrent in pyramidal cells that express both HCN and Slack channels, indicating that blockade of HCN channels reduced K+current indirectly by lowering Na+influx. In contrast, ZD7288 has no effect on KNacurrents in an HEK cell line stably expressing this Slack channels but no HCN channels, demonstrating that ZD7288 does not block Slack channels directly. Activation of HCN channels by cAMP in a cell line expressing a Ca2+reporter results in elevation of cytoplasmic Ca2+, but the effect of cAMP is completely reversed if the HCN channels are co-expressed with Slack channels. Finally, we have used a novel pharmacological blocker of Slack channels to show that inhibition of either Slack or HCN channels in rat PFC improves working memory performance, and that the actions of Slack and HCN channel blockers occlude each other in the memory task. Our results suggest that the regulation of working memory by HCN channels in PFC pyramidal neurons is mediated by an HCN-Slack channel complex that links activation HCN channels to suppression of neuronal excitability.