KCC3-dependent chloride extrusion in adult sensory neurons

Abstract

The cation-Cl(-) cotransporters participate to neuronal Cl(-) balance and are responsible for the post-natal Cl(-) switch in central neurons. In the adult peripheral nervous system, it is not well established whether a Cl(-) transition occurs during maturation. We investigated the contribution of cation-Cl(-) cotransporters in the Cl(-) handling of sensory neurons derived from the dorsal root ganglia (DRG) of neonatal mice (postnatal days 1-6) and adult mice. Gramicidin-perforated patch-clamp recordings in wild-type neurons revealed that Cl(-) accumulated to very high values in P1-6 sensory neurons and decreased in adulthood. In post-natal sensory neurons, quantitative RT-PCR showed that NKCC1, KCC1 and KCC3 had a higher transcript expression level compared to KCC2 and KCC4. NKCC1 was the main cation-Cl(-) cotransporter controlling Cl(-) accumulation at this developmental stage. In adulthood, the KCC3 transcript was produced in larger amounts than the other cation-Cl(-) cotransporter transcripts and RT-PCR shows larger expression of the shorter KCC3a isoform in adult DRG. Pharmacological inhibitors of cation-Cl(-) cotransporters and the use of KCC3(-/-) mice demonstrated that NKCC1 sustained Cl(-) accumulation in the majority of adult sensory neurons while KCC3 contributed to Cl(-) extrusion in a subset of these neurons. Beta-galactosidase detection in adult KCC3(-/-) DRG showed that KCC3 transcripts were present in all adult sensory neurons suggesting a KCC3 isoform specific regulation of Cl(-) handling. The contribution of KCC3 to Cl(-) extrusion in a subset of sensory neurons indicates that KCC3 could play a major role in GABAergic/glycinergic transmission.