The Na+/H+ exchangers of intracellular compartments. Biochemical and pharmacological characterization. Physiological roles and impact in neurological diseases. – NHEINT

The acidification of intracellular compartments is critical for a wide range of cellular processes, such as protein maturation, degradation, receptor recycling and desensitization as well as neurotransmitter loading in synaptic vesicles.
This acidification occurs through a steady-state mechanism that maintains stable vesicular pH values by coupling a constitutive proton loading to a regulable proton leak. The loading mechanism consists in a proton ATPase, coupled to ClC chloride transporters which counterbalance the influx of H+ positive charges. By contrast, the molecular identity of the proton leak has never been clearly determined. Three conserved isoforms of Na+/H+ exchangers that encode transporters localized in intracellular compartments have been cloned in the past years. Their mRNAs have been detected in most cells and tissues. Due to the ion gradients between the lumen of the vesicles and the cytoplasm, these exchangers must thermodynamically extrude protons into the cytoplasm and are extremely good candidates for the above-described proton leak system.
Despite this potential importance, these transporters have raised limited attention. However, in 2006, a NHE-7 gene deletion has been associated to mental retardation and in 2008, two studies have linked defects of NHE-6 and NHE-9 with genetic syndromes that include autism-like cognitive disorders, epilepsy and cerebellum degeneration. This highlights the importance of these transporters and suggests that steady state regulation of the pH of intracellular compartments is involved in brain plasticity and cognitive functions, possibly through protein turnover, receptor desensitization or neurotransmitter loading and release. Based on their importance and due to the scarcity of data on the functional parameters and roles of these intracellular transporters, we propose to use our expertise in the field of Na+/H+ exchangers and intracellular ion transporters to study them in detail. The aims and parts of our proposal are summarized as follows:
(i) The investigation of the biochemical and pharmacological features of the intracellular Na+/H+ exchangers NHE-6, 7 and 9. This is problematic because their intracellular targeting limits their accessibility. To solve this problem, our laboratory has developed unique specific somatic cell genetic techniques that enable the selection of variant cell lines able to resist H+ killing through plasma membrane expression of H+ extruders. We have successfully used these techniques to obtain the stable cell lines with forced plasma membrane expression of NHE-6, 7 and 9. We can now apply the fast transport measurements that we have developed to study their kinetics, ion selectivity, inhibitors and regulation in an almost exhaustive manner.
(ii) The use the NHE-6 deficient mouse model to study how the disfunction of such a transporter produces neurological phenotypes
(iii) We propose to initiate a new pharmacological approach aimed at compensating the progressive loss of acidification of intracellular compartment that occurs with aging or following ischemia reperfusion. This results in decreased activity of acidic proteases and in the accumulation of undegraded material (ceroïd lipofuscinosis) that impairs the function of postmitotic cells (brain, heart, kidney). We plan to use the knowledge gained in (i) to construct here a series of benzoguanidine-derived inhibitors specific of the intracellular NHEs that will accumulate intracellularly and reduce the proton leak form acidic compartments. As it is not possible to pharmacologically re-activate the H+ load, we think that an approach aimed at reducing the leak could constitute a very promising strategy.
(iv) Finally we plan to search for new mutations in NHEint genes in human neurological diseases and study their effects on the protein function. This will be done in collaboration with clinicians working with young patients exhibiting cognitive disorders associated with seizures.