Putative binding sites for benzocaine on a human cardiac cloned channel (Kv1.5).
OBJECTIVE
It has been demonstrated that at nanomolar concentrations benzocaine increased, whereas at micromolar concentrations, it blocked hKv1.5 channels in a voltage-dependent manner and modified the voltage-dependence of channel activation. The present study was undertaken to localize the putative binding sites involved in the 'agonists' and blocking effects of benzocaine.


METHODS
Experiments were carried out on wild-type and site directed mutated hKv1.5 channels stably expressed on Ltk(-) cells using the whole-cell patch-clamp.


RESULTS
At 35 mM [K+](i) the voltage-dependent unblock produced by 500 microM benzocaine was preserved at both 4 and 140 mM [K+](o). Mutations located in the inner mouth of the pore (T477S, T505A, L508M and V512M) abolished the agonist but increased the blocking effects of benzocaine. Intracellular application of tetraethylammonium (3 mM) abolished the 'agonist' effects whereas the blocking effects of benzocaine remained unaltered. Block induced by benzocaine and intracellular tetraethylammonium was additive. In contrast, the combination of benzocaine and bupivacaine (>25 microM) produced less blockade than bupivacaine alone. However, mutation of the extracellular residue R485Y did not modify the effects of benzocaine. Extracellular application of tetraethylammonium (100 mM) did not modify the agonist effects of benzocaine, but abolished the voltage- and time-dependence of benzocaine-induced block.


CONCLUSIONS
The results suggested that benzocaine binds with high affinity to an intracellular binding site to produce 'agonist' effects and to a low affinity subsite, which is also located in the inner mouth, to produce the blocking effects. Furthermore, benzocaine and extracellular K(+) interact to modify the voltage-dependence of channel opening.
Caballero
Ricardo
R
Department of Pharmacology, School of Medicine, Universidad Complutense, Madrid, Spain. rcaballero@ift.csic.es
Moreno
Ignacio
I
González
Teresa
T
Valenzuela
Carmen
C
Tamargo
Juan
J
Delpón
Eva
E
D000818:Animals; D001566:Benzocaine / Q000378:metabolism*; D001665:Binding Sites; D002045:Bupivacaine / Q000494:pharmacology; D002460:Cell Line; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects*; D051666:Kv1.5 Potassium Channel; D051379:Mice; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D019789:Tetraethylammonium / Q000494:pharmacology; D014162:Transfection
C496981:KCNA5 protein, human; C496982:Kcna5 protein, mouse; D051666:Kv1.5 Potassium Channel; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D019789:Tetraethylammonium; D011188:Potassium; D001566:Benzocaine; D002045:Bupivacaine