Time-dependent block and resurgent tail currents induced by mouse beta4(154-167) peptide in cardiac Na+ channels.
Resurgent tail Na(+) currents were first discovered in cerebellar Purkinje neurons. A recent study showed that a 14-mer fragment of a mouse beta4 subunit, beta4(154-167), acts as an intracellular open-channel blocker and elicits resurgent currents in Purkinje neurons (Grieco, T.M., J.D. Malhotra, C. Chen, L.L. Isom, and I.M. Raman. 2005. Neuron. 45:233-244). To explore these phenotypes in vitro, we characterized beta4(154-167) actions in inactivation-deficient cardiac hNav1.5 Na(+) channels expressed in human embryonic kidney 293t cells. Intracellular beta4(154-167) from 25-250 microM elicited a conspicuous time-dependent block of inactivation-deficient Na(+) currents at 50 mV in a concentration-dependent manner. On and off rates for beta4(154-167) binding were estimated at 10.1 microM(-1)s(-1) and 49.1 s(-1), respectively. Upon repolarization, large tail currents emerged with a slight delay at -140 mV, probably as a result of the rapid unblocking of beta4(154-167). Near the activation threshold (approximately -70 mV), resurgent tail currents were robust and long lasting. Likewise, beta4(154-167) induces resurgent currents in wild-type hNav1.5 Na(+) channels, although to a lesser extent. The inactivation peptide acetyl-KIFMK-amide not only restored the fast inactivation phenotype in hNav1.5 inactivation-deficient Na(+) channels but also elicited robust resurgent currents. When modified by batrachotoxin (BTX), wild-type hNav1.5 Na(+) channels opened persistently but became resistant to beta4(154-167) and acetyl-KIFMK-amide block. Finally, a lysine substitution of a phenylalanine residue at D4S6, F1760, which forms a part of receptors for local anesthetics and BTX, rendered cardiac Na(+) channels resistant to beta4(154-167). Together, our in vitro studies identify a putative S6-binding site for beta4(154-167) within the inner cavity of hNav1.5 Na(+) channels. Such an S6 receptor readily explains (1) why beta4(154-167) gains access to its receptor as an open-channel blocker, (2), why bound beta4(154-167) briefly prevents the activation gate from closing by a "foot-in-the-door" mechanism during deactivation, (3) why BTX inhibits beta4(154-167) binding by physical exclusion, and (4) why a lysine substitution of residue F1760 eliminates beta4(154-167) binding.
Wang
Ging Kuo
GK
Department of Anesthesia, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115, USA. wang@zeus.bwh.harvard.edu
Edrich
Thomas
T
Wang
Sho-Ya
SY
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D009124:Muscle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D062554:NAV1.5 Voltage-Gated Sodium Channel; D010455:Peptides / Q000008:administration & dosage*; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013997:Time Factors
D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D010455:Peptides; C568320:SCN5A protein, human; C568321:Scn5a protein, mouse; D015222:Sodium Channels