Nanna K. Jorgensen

KCNE4 is an inhibitory subunit to the KCNQ1 channel

KCNE4 is a membrane protein belonging to a family of single transmembrane domain proteins known to have dramatic effect on the gating of certain potassium channels. However, no functional role of KCNE4 has been suggested so far. In the present paper we demonstrate that KCNE4 is an inhibitory subunit to KCNQ1 channels. Co‐expression of KCNQ1 and KCNE4 in Xenopus oocytes completely inhibited the KCNQ1 current. This was reproduced in mammalian CHO‐K1 cells. Experiments with delayed expression of mRNA coding for KCNE4 in KCNQ1‐expressing oocytes suggested that KCNE4 exerts its effect on KCNQ1 channels already expressed in the plasma membrane. This notion was supported by immunocytochemical studies and Western blotting, showing no significant difference in plasma membrane expression of KCNQ1 channels in the presence or absence of KCNE4. The impact of KCNE4 on KCNQ1 was specific since no effect of KCNE4 could be detected if co‐expressed with KCNQ2‐5 channels or hERG1 channels. RT‐PCR studies revealed high KCNE4 expression in embryos and adult uterus, where significant expression of KCNQ1 channels has also been demonstrated.

Requirement of subunit co-assembly and ankyrin-G for M-channel localization at the axon initial segment

The potassium channel subunits KCNQ2 and KCNQ3 are believed to underlie the M current of hippocampal neurons. The M-type potassium current plays a key role in the regulation of neuronal excitability; however, the subcellular location of the ion channels underlying this regulation has been controversial. We report here that KCNQ2 and KCNQ3 subunits are localized to the axon initial segment of pyramidal neurons of adult rat hippocampus and in cultured hippocampal neurons. We demonstrate that the localization of the KCNQ2/3 channel complex to the axon initial segment is favored by co-expression of the two channel subunits. Deletion of the ankyrin-G-binding motif in both the KCNQ2 and KCNQ3 C-terminals leads to the disappearance of the complex from the axon initial segment, albeit the channel complex remains functional and still reaches the plasma membrane. We further show that although heteromeric assembly of the channel complex favours localization to the axon initial segment, deletion of the ankyrin-G-binding motif in KCNQ2 alone does not alter the subcellular localization of KCNQ2/3 heteromers. By contrast, deletion of the ankyrin-G-binding motif in KCNQ3 significantly reduces AIS enrichment of the complex, implicating KCNQ3 as a major determinant of M channel localization to the AIS.

KCNQ1 channels sense small changes in cell volume.

Many important physiological processes involve changes in cell volume, e.g. the transport of salt and water in epithelial cells and the contraction of cardiomyocytes. In this study, we show that voltage‐gated KCNQ1 channels, which are strongly expressed in epithelial cells or cardiomyocytes, and KCNQ4 channels, expressed in hair cells and the auditory tract, are tightly regulated by small cell volume changes when co‐expressed with aquaporin 1 water‐channels (AQP1) in Xenopus oocytes. The KCNQ1 and KCNQ4 current amplitudes precisely reflect the volume of the oocytes. By contrast, the related KCNQ2 and KCNQ3 channels, which are prominently expressed in neurons, are insensitive to cell volume changes. The sensitivity of the KCNQ1 and KCNQ4 channels to cell volume changes is independent of the presence of the auxiliary KCNE1–3 subunits, although modulated by KCNE1 in the case of KCNQ1. Incubation of the oocytes in cytochalasin D and experiments with truncated KCNQ1 channels suggest that KCNQ1 channels sense cell volume changes through interactions between the cytoskeleton and the N‐terminus of the channel protein. From our results we propose that KCNQ1 and KCNQ4 channels play an important role in cell volume control, e.g. during transepithelial transport of salt and water.

Basolateral localisation of KCNQ1 potassium channels in MDCK cells: molecular identification of an N-terminal targeting motif

KCNQ1 potassium channels are expressed in many epithelial tissues as well as in the heart. In epithelia KCNQ1 channels play an important role in salt and water transport and the channel has been reported to be located apically in some cell types and basolaterally in others. Here we show that KCNQ1 channels are located basolaterally when expressed in polarised MDCK cells. The basolateral localisation of KCNQ1 is not affected by co-expression of any of the five KCNE β-subunits. We characterise two independent basolateral sorting signals present in the N-terminal tail of KCNQ1. Mutation of the tyrosine residue at position 51 resulted in a non-polarized steady-state distribution of the channel. The importance of tyrosine 51 in basolateral localisation was emphasized by the fact that a short peptide comprising this tyrosine was able to redirect the p75 neurotrophin receptor, an otherwise apically located protein, to the basolateral plasma membrane. Furthermore, a di-leucine-like motif at residues 38-40 (LEL) was found to affect the basolateral localisation of KCNQ1. Mutation of these two leucines resulted in a primarily intracellular localisation of the channel.

Mechanisms of pHi regulation studied in individual neurons cultured from mouse cerebral cortex

Maintenance and regulation of intracellular pH (pHi) was studied in single cultured mouse neocortical neurons using the fluorescent probe 2′,7′‐bis‐(2‐carboxyethyl)‐5,6‐carboxyfluorescein (BCECF). Reversal of the Na+ gradient by reduction of the extracellular Na+ concentration ([Na+]o) resulted in rapid intracellular acidification, inhibited by 5′‐(N‐ethyl‐N‐isopropyl)amiloride (EIPA), an inhibitor of Na+/H+ exchange. In the presence of EIPA and/or 4′,4′‐diisothiocyano‐stilbene‐2′,2′‐sulfonic acid (DIDS), an inhibitor of Na+‐coupled anion exchangers and Na+‐HCO3− cotransport, a slow decline of pHi was seen. Following intracellular acidification imposed by an NH4Cl prepulse, pHi recovered at a rapid rate, which was reduced by reduction of [Na+]o and was virtually abolished by EIPA and DIDS in combination. Creating an outward Cl− gradient by removal of extracellular Cl− significantly increased the rate of pHi recovery. In HCO3−‐free media, the pHi recovery rate was reduced in control cells and was abolished at zero [Na+]o and by EIPA. After intracellular alkalinization imposed by an acetate prepulse, pHi recovery was unaffected by DIDS but was significantly reduced in the absence of extracellular Cl−, as well as in the presence of Zn2+, which is a blocker of proton channels. Together, this points toward a combined role of DIDS‐insensitive Cl−/HCO3− and passive H+ influx in the recovery of pHi after alkalinization. J. Neurosci. Res. 51:431–441, 1998. © 1998 Wiley‐Liss, Inc.

KCNQ Channels are Sensors of Cell Volume

Many important physiological processes involve changes in cell volume, e.g., the transport of salt and water in epithelial cells and the contraction of muscle cells. These cells respond to swelling with a so-called regulatory volume decrease which involves the activation of K+ channels. However, the molecular identity of the involved K+ channels has not been clear, and in particular, the mechanism for activation has been obscure.