Modulation of the cardiac sodium channel NaV1.5 by Fyn, a Src family tyrosine kinase.
Dynamic modulation of ion channels can produce dramatic alterations of electrical excitability in cardiac myocytes. This study addresses the effects of the Src family tyrosine kinase Fyn on Na(V)1.5 cardiac sodium channels. Sodium currents were acquired by whole cell recording on HEK-293 cells transiently expressing Na(V)1.5. Acute treatment of cells with insulin caused a depolarizing shift in steady-state inactivation, an effect eliminated by the Src-specific tyrosine kinase inhibitor PP2. Sodium channels were coexpressed with either constitutively active (Fyn(CA)) or catalytically inactive (Fyn(KD)) variants of Fyn. Fyn(CA) caused a 10-mV depolarizing shift of steady-state inactivation compared with Fyn(KD) without altering the activation conductance-voltage relationship. Comparable effects of these Fyn variants were obtained with whole-cell and perforated-patch recording. Tyrosine phosphorylation of immunoprecipitated Na(V)1.5 was increased in cells expressing Fyn(CA) compared with Fyn(KD). We show that Fyn is present in rat cardiac myocytes, and that Na(V)1.5 channels from these myocytes are tyrosine-phosphorylated. In HEK-293 cells the effect of Fyn(CA) on Na(V)1.5 inactivation is abolished by the single point mutation Y1495F, a residue located within the cytoplasmic linker between the third and fourth homologous domains of the sodium channel. We provide evidence that this linker is a substrate for Fyn in vitro, and that Y1495 is a preferred phosphorylation site. These results suggest that cardiac sodium channels are physiologically relevant targets of Src family tyrosine kinases.
Ahern
Christopher A
CA
Department of Physiology, Institute of Hyperexcitability, Jefferson Medical College, Philadelphia, Pa 19107, USA.
Zhang
Ji-Fang
JF
Wookalis
Marilyn J
MJ
Horn
Richard
R
D000818:Animals; D002460:Cell Line; D004553:Electric Conductivity; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D062554:NAV1.5 Voltage-Gated Sodium Channel; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011506:Proteins / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D051381:Rats; D015222:Sodium Channels / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*; D000097847:Protein Phosphatase Inhibitory Proteins
D007328:Insulin; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D011506:Proteins; D011518:Proto-Oncogene Proteins; D051076:Proto-Oncogene Proteins c-fyn; D015222:Sodium Channels; D014443:Tyrosine; D019061:src-Family Kinases; C568320:SCN5A protein, human; C568322:Scn5a protein, rat; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins; C494957:FYN protein, human; C494961:Fyn protein, rat