Three Mechanisms Underlie KCNQ2/3 Heteromeric Potassium M-Channel Potentiation

Abstract

The non-inactivating potassium M-current exerts a strong influence on neuronal excitability. The channels responsible for this current are made up of KCNQ subunits, and mutations in most of these produce human pathologies. Notably, in terms of excitation, mutations in either KCNQ2 or KCNQ3 lead to benign neonatal familial convulsions. Although a mere reduction of 25% in KCNQ2/3 function can increase excitability to epileptogenic levels, the potentiation of these subunits has anti-epileptogenic effects. After KCNQ2/3 heteromerization, current levels can augment as much as 10-fold, and we have discovered that there are three processes underlying this potentiation. First, there is an increase in the number of channels inserted in the membrane after heteromerization of the C-terminal region. Second, the N-terminal domain from KCNQ2 exerts a negative influence on the current level. Finally, Ala 315 of KCNQ3, a residue located in the inner vestibule after the selectivity filter, plays a critical role in preventing current flow in KCNQ3 homomeric channels, whereas it is permissive in heteromers in combination with Thr at the equivalent 276 position of KCNQ2.

FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization

“…According to these results, it seems that interaction between PIP1s and PIP2s could be a means of regulating their activity, as observed for several other membrane proteins for which multimerization is an important regulation mechanism (17)(18)(19). Because this interaction of plant PIP1s and PIP2s was observed in heterologous expression systems (Xenopus oocytes or COS cells) with different water requirements and protein regulations, it is essential to determine whether such interactions occur in plant cells.…”

“…The suspension was then mixed with 110 l of PEG medium (40% PEG 4000/0.1 M Ca(NO 3 ) 2 /0.3 M mannitol) and incubated for 2 min at room temperature (RT). After the addition of 440 l of W5 medium (154 mM NaCl/125 mM CaCl 2 /5 mM KCl/4 mM Mes, pH 5.7), the protoplasts were collected by centrifugation at 110 ϫ g for 1 min, resuspended in 1.1 ml of incubation buffer (0.6 M mannitol/4 mM KCl/4 mM Mes, pH 5.7), and kept in the dark at 20°C for [16][17][18][19][20][21][22][23][24] h in the presence of 90 g/ml cefotaxime. RNA extraction and quantitative RT-PCR were performed as described previously (31).…”

Lack of correlation between surface expression and currents in epileptogenic AB-calmodulin binding domain Kv7.2 potassium channel mutants

“…To investigate the trafficking of the channels to the plasma membrane, Kv7.3 subunits tagged with an HA epitope at an extracellular loop were used as reporter for surface expression in Xenopus oocytes [15,31,32]. Consistent with the reduction in current amplitude, R333Q mutant surface expression was diminished (~ 50%).…”

“…Xenopus oocytes preparation and standard two-electrode voltage clamp were performed as previously described [31,47]. Stage V or VI oocytes were defolliculated with 1 mg/ml collagenase (C9891; Sigma) in Ca 2+ -free OR2 (5 mM HEPES, 82.5 mM NaCl, 2.5 mM KCl and 1 mM MgCl 2 , pH 7.5), and then were transferred to a Ca 2+ -containing solution ND96 (5 mM HEPES, 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , pH 7.5).…”

“…We followed the method based on luminescence described by Schwake and coworkers using the Kv7.3 subunit tagged with a hemagglutinin (HA) epitope (YPYDVPDYA) in the extracellular loop that connects transmembrane domains S1 and S2 [31,32]. Oocytes were injected with 20 ng of a 1:1 cRNA mixture that always included 10 ng of Kv7.3-HA.…”