ABSTRACT
Title
Gating currents from neuronal Kv7 channels carrying BFNS-causing mutations in the S4 segment of the Voltage Sensing Domain
Authors
F. Miceli 1,2, E. Vargas 3, MR. Cilio 1, F. Bezanilla 3 and M. Taglialatela 2,4
1Div. Neurology, IRCCS Bambino Gesù Children's Hosp, Rome, Italy; 2Dept. Neuroscience, University of Naples "Federico II", Naples, Italy; 3Dept. of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA; 4Dept. of Health Science, University of Molise, Campobasso, Italy;
1Div. Neurology, IRCCS Bambino Gesù Children's Hosp, Rome, Italy; 2Dept. Neuroscience, University of Naples "Federico II", Naples, Italy; 3Dept. of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA; 4Dept. of Health Science, University of Molise, Campobasso, Italy;
Abstract
Among voltage-dependent K+channels (Kv), the Kv7 family is composed of 5 members (Kv7.1-Kv7.5), each showing a different tissue distribution and distinct physiological role (Miceli et al., 2008). Mutations in four of the five genes coding for Kv7 channels have been linked to different human channelopathies. Mutations in Kv7.1 cause one form of long QT syndrome, whereas Kv7.4 mutations underlie a rare form of slowly progressive deafness (DFNA2); finally, mutations in Kv7.2/Kv7.3 genes have been identified in families affected by Benign Familial Neonatal Seizures (BFNS) and/or peripheral nerve hyperexcitability (PNH). Of these hereditary diseases, BFNS represent a rare form of idiopathic epilepsy with autosomal-dominant transmission characterized by the onset of focal, multifocal or generalized tonic-clonic seizures around the third day of post-natal life that disappear after few weeks or months. Drugs acting as openers of Kv7 channels are under close scrutiny for their potential anticonvulsant effects in humans (Miceli et al, 2008; Barrese et al., 2010).
Structurally, Kv channels are homo-or hetero-tetramers formed by the assembly of identical or compatible subunits, respectively. Each subunit is composed of six transmembrane segments (S1-S6); the S5 and S6 segments and the intervening linker form the K+-selective aqueous pore and the inner pore gate, whereas the S1-S4 region forms the Voltage Sensing Domain (VSD), a functional domain responsible for voltage-sensitivity. Disease-causing mutations often affect residues in the VSD of Kv7 subunits and modify the gating properties of the macroscopic currents carried by these channels. To achieve a more detailed functional analysis of these gating changes, using the cut-open vaseline gap technique we recorded the gating currents from neuronal Kv7.4 channels (Miceli et al., 2009). Gating currents are transient currents generated by the displacement of charged elements within the VSD in response to changes in transmembrane voltage; they represent a direct measurement of the charge displacementoccurring during VSD movement within the phospholipid bilayer of the cell membrane. In the present work, we have characterized the ionic and gating currents from homomeric Kv7.4 channels carrying mutations homologous to BFNS-causing mutations in Kv7.2 (R213Q/W, D218G and R219W), as well as the non-BFNS mutant R219Q (Kv7.4 numbering).
Channels carrying the D218G, R219Q or the R219W mutations in the C-terminal part of S4 show the following salient properties: +30 mV shift of the G/V curves and faster activation/deactivation kinetics. Gating currents from these channels showed: gating charge conservation between QONand QOFF, slight (5-13 mV) right-shift of the Q/V curves, and a fast QOFFdecay with no rising phase.
The slight shift of Q/V curves compared to the marked shift of G/V curves observed suggests that the D218G, R219Q and R219W mutations affect mainly the coupling of the VSD movement to pore opening rather than the voltage-sensitivity of the VSD movement. Moreover, the fast QOFF decay with no rising phase observed in these mutants may correlate with the stabilization of the closed conformation of the channel. Neutralization of R213 caused a G/V right-shift associated to slower activation/deactivation channel kinetics. Interestingly, upon pore current blockade with TEA and Ba2+, in Kv7.4 channels carrying the R213Q/W mutations causing PNH,a persistent outward current was recorded that increases on depolarization, indicating a gating pore current in the active state of the VSD, thus revealing a novel potentially-relevant pathophysiological mechanism for PHN in humans.
Supported by Telethon GP07125 (MT), Fondazione San Paolo (Programma Neuroscienze 2008) (MT), E-Rare JTC 2007 (MRC), and NIH GM30376 (FB).
Miceli et al., (2008). Curr Opin Pharmacol. 8, 65-74.
Miceli et al., (2009). Channels. 3, 274-83.
Barrese et al., (2010) Clinical Pharmacology: Advances and Applications. 2, 225-336
Structurally, Kv channels are homo-or hetero-tetramers formed by the assembly of identical or compatible subunits, respectively. Each subunit is composed of six transmembrane segments (S1-S6); the S5 and S6 segments and the intervening linker form the K+-selective aqueous pore and the inner pore gate, whereas the S1-S4 region forms the Voltage Sensing Domain (VSD), a functional domain responsible for voltage-sensitivity. Disease-causing mutations often affect residues in the VSD of Kv7 subunits and modify the gating properties of the macroscopic currents carried by these channels. To achieve a more detailed functional analysis of these gating changes, using the cut-open vaseline gap technique we recorded the gating currents from neuronal Kv7.4 channels (Miceli et al., 2009). Gating currents are transient currents generated by the displacement of charged elements within the VSD in response to changes in transmembrane voltage; they represent a direct measurement of the charge displacementoccurring during VSD movement within the phospholipid bilayer of the cell membrane. In the present work, we have characterized the ionic and gating currents from homomeric Kv7.4 channels carrying mutations homologous to BFNS-causing mutations in Kv7.2 (R213Q/W, D218G and R219W), as well as the non-BFNS mutant R219Q (Kv7.4 numbering).
Channels carrying the D218G, R219Q or the R219W mutations in the C-terminal part of S4 show the following salient properties: +30 mV shift of the G/V curves and faster activation/deactivation kinetics. Gating currents from these channels showed: gating charge conservation between QONand QOFF, slight (5-13 mV) right-shift of the Q/V curves, and a fast QOFFdecay with no rising phase.
The slight shift of Q/V curves compared to the marked shift of G/V curves observed suggests that the D218G, R219Q and R219W mutations affect mainly the coupling of the VSD movement to pore opening rather than the voltage-sensitivity of the VSD movement. Moreover, the fast QOFF decay with no rising phase observed in these mutants may correlate with the stabilization of the closed conformation of the channel. Neutralization of R213 caused a G/V right-shift associated to slower activation/deactivation channel kinetics. Interestingly, upon pore current blockade with TEA and Ba2+, in Kv7.4 channels carrying the R213Q/W mutations causing PNH,a persistent outward current was recorded that increases on depolarization, indicating a gating pore current in the active state of the VSD, thus revealing a novel potentially-relevant pathophysiological mechanism for PHN in humans.
Supported by Telethon GP07125 (MT), Fondazione San Paolo (Programma Neuroscienze 2008) (MT), E-Rare JTC 2007 (MRC), and NIH GM30376 (FB).
Miceli et al., (2008). Curr Opin Pharmacol. 8, 65-74.
Miceli et al., (2009). Channels. 3, 274-83.
Barrese et al., (2010) Clinical Pharmacology: Advances and Applications. 2, 225-336