The physiological role of potassium-chloride cotransporters (KCCs) in the cerebellum

Abstract

Cation chloride cotransporters mediate a coupled electroneutral movement of Cl-, K+ and Na+ across plasmamembranes in many cells. The neuron-specific KCl cotransporter KCC2 is thought to lower the intracellular Cl- concentration below its electrochemical equilibrium potential by using the outwards directed gradient of K+ as a driving force. This low intracellular Cl- concentration is required for the fast inhibitory action of GABA which is mediated by the GABAA receptor, a ligand-gated anion channel. The activation of GABAA receptors drives the membrane potential of a cell towards EGABA, the reversal potential of GABAergic currents. In immature neurons, GABA is excitatory, as EGABA is above the resting membrane potential. The expression of KCC2 correlates with the drop of EGABA below the resting membrane potential, and thereby the switch from excitatory to inhibitory GABA signaling. The KCl cotransporter KCC3 is expressed more broadly and involved in cell volume regulation. Nevertheless, while KCC2 may be the key regulator of neuronal [Cl-]i, a similar role was proposed for KCC3. To investigate the physiological role of KCC2 and KCC3 in the murine cerebellum at cellular level, we specifically delete the KCCs in cerebellar Purkinje cells and Granule cells by breeding floxed mice with alpha6-Cre- and L7-Cre-mice, respectively. To investigate a possible role for GABAergic inhibition on the cellular level we record from acute slices with the perforated patch technique. We showed that GABAergic currents of wildtype Purkinje cells (>P25) reverse around -95 mV. For KCC2-/- Purkinje cells this reversion is shifted to more positive potentials, but still remains hyperpolarising. Granule cells of wildtype mice show a strong membrane staining for KCC2. Surprisingly, the driving force for Cl- in respect to the resting membrane potential was not altered in granule cells lacking KCC2. It would be feasible to examine the cerebellar circuit by stimulating parallel fibers while recording from Purkinje cells. The feed forward inhibition in this circuitry should be altered in knockout mice. These experiments might give answers to the question how the cerebellar network is processing information