SK2-containing channels are expressed in the postsynaptic density (PSD) of dendritic spines on mouse CA1 pyramidal neurons, and influence synaptic responses, plasticity, and learning. The SK2 gene encodes two isoforms differing only in the length of the N-terminal domain. SK2-Long (SK2-L) and SK2-Short (SK2-S) are co-expressed in CA1 pyramidal neurons and likely form heteromeric channels. In mice lacking SK2-L (SK2-Sonly mice), SK2-S-containing channels were expressed in the extrasynaptic membrane, but were excluded from the PSD. The SK channel contribution to EPSPs was absent in SK2-Sonly mice, and was restored by SK2-L re-expression. In slices from wild type mice, blocking SK channels increased the amount of long-term potentiation (LTP) induced in area CA1 but was without effect in SK2-Sonly mice. Further, SK2-Sonly mice outperformed wild type mice in the novel object recognition task. These results show that SK2-L directs synaptic SK2-containing channel expression, important for normal synaptic signaling, plasticity, and learning.

Several reports have described excitatory GABA transmission in the suprachiasmatic nucleus (SCN), the master pacemaker of circadian physiology. However, there is disagreement regarding the prevalence, timing, and neuronal location of excitatory GABA transmission in the SCN. Whether GABA is inhibitory or excitatory depends, in part, on the intracellular concentration of chloride ([Cl−]i). Here, using ratiometric Cl− imaging, we have investigated intracellular chloride regulation in AVP and VIP-expressing SCN neurons and found evidence suggesting that [Cl−]i is higher during the day than during the night in both AVP+ and VIP+ neurons. We then investigated the contribution of the cation chloride cotransporters to setting [Cl−]i in these SCN neurons and found that the chloride uptake transporter NKCC1 contributes to [Cl−]i regulation in SCN neurons, but that the KCCs are the primary regulators of [Cl−]i in SCN neurons. Interestingly, we observed that [Cl−]i is differentially regulated between AVP+ and VIP+ neurons-a low concentration of the loop diuretic bumetanide had differential effects on AVP+ and VIP+ neurons, while blocking the KCCs with VU0240551 had a larger effect on VIP+ neurons compared to AVP+ neurons.

Background and purpose: Neuronal ion channels are key targets of general anaesthetics and alcohol, and binding of these drugs to pre-existing and relatively specific sites is thought to alter channel gating. However, the underlying molecular mechanisms of this action are still poorly understood. Here, we investigated the neuronal Shaw2 voltage-gated K + (Kv) channel to ask whether the inhalational anaesthetic halothane and n-alcohols share a binding site near the activation gate of the channel. Experimental approach: Focusing on activation gate mutations that affect channel modulation by n-alcohols, we investigated n-alcohol-sensitive and n-alcohol-resistant Kv channels heterologously expressed in Xenopus oocytes to probe the functional modulation by externally applied halothane using two-electrode voltage clamping and a gas-tight perfusion system. Key results: Shaw2 Kv channels are reversibly inhibited by halothane in a dose-dependent and saturable manner (K0.5 = 400 mM; nH = 1.2). Also, discrete mutations in the channel's S4S5 linker are sufficient to reduce or confer inhibition by halothane (Shaw2-T330L and Kv3.4-G371I/T378A respectively). Furthermore, a point mutation in the S6 segment of Shaw2 (P410A) converted the halothane-induced inhibition into halothane-induced potentiation. Lastly, the inhibition resulting from the co-application of n-butanol and halothane is consistent with the presence of overlapping binding sites for these drugs and weak binding cooperativity. Conclusions and implications:These observations strongly support a molecular model of a general anaesthetic binding site in the Shaw2 Kv channel. This site may involve the amphiphilic interface between the S4S5 linker and the S6 segment, which plays a pivotal role in Kv channel activation.

Cl--sensitive with-no-lysine kinase (WNK) plays a key role in regulating the thiazide-sensitive sodium-chloride cotransporter (NCC) in the distal convoluted tubule (DCT). Cl- enters DCT cells through NCC and leaves the cell across the basolateral membrane via the Cl- channel ClC-Kb or a K-Cl cotransporter (KCC). While KCC is electroneutral, Cl- exit via ClC-Kb is electrogenic. Therefore, an alteration in DCT basolateral K+ channel activity is expected to influence Cl- movement across the basolateral membrane. Although a role for intracellular Cl- in the regulation of WNK and NCC has been established, intracellular Cl- concentrations ([Cl-]i) have not been directly measured in mammalian DCT. Therefore, to measure [Cl-]i in DCT cells, we have generated a transgenic mouse model expressing an optogenetic kidney-specific Cl-Sensor and measured Cl- fluorescent imaging in the isolated DCT. Basal measurements indicate that the mean [Cl-]i is approximately 7 mM. Stimulation of chloride exit with low chloride hypotonic solutions decreased [Cl-]i, whereas inhibition of KCC by DIOA or inhibition of ClC-Kb by NPPB increased [Cl-]i, suggesting roles for both KCC and ClC-Kb modulating [Cl-]i . Blocking the basolateral K+ channels, Kir4.1/5.1, with barium significantly increased the [Cl-]i. Finally, a decrease in extracellular K+ concentration transiently decreased [Cl-]i whereas raising extracellular K+ transiently increased [Cl-]i, further suggesting a role for Kir4.1/5.1 in the regulation of [Cl-]i. We conclude that the alteration in ClC-Kb, KCC and Kir4.1/5.1 activity influences the [Cl-]i in the DCT.