Characterization of the human HCN1 channel and its inhibition by capsazepine.
The human hyperpolarization-activated cyclic nucleotide-gated 1 (hHCN1) subunit was heterologously expressed in mammalian cell lines (CV-1 and CHO) and its properties investigated using whole-cell patch-clamp recordings. Activation of this recombinant channel, by membrane hyperpolarization, generated a slowly activating, noninactivating inward current. The pharmacological properties of hHCN1-mediated currents resembled those of native hyperpolarization-activated currents (I(h)), that is, blockade by Cs(+) (99% at 5 mm), ZD 7288 (98% at 100 microm) and zatebradine (92% at 10 microm). Inhibition of the hHCN1-mediated current by ZD 7288 was apparently independent of prior channel activation (i.e. non-use-dependent), whereas that induced by zatebradine was use-dependent. The VR1 receptor antagonist capsazepine inhibited hHCN1-mediated currents in a concentration-dependent (IC(50)=8 microm), reversible and apparently non-use-dependent manner. This inhibitory effect of capsazepine was voltage-independent and associated with a leftward shift in the hHCN1 activation curve as well as a dramatic slowing of the kinetics of current activation. Elevation of intracellular cAMP or extracellular K(+) significantly enhanced aspects of hHCN1 currents. However, these manipulations did not significantly affect the capsazepine-induced inhibition of hHCN1. The development of structural analogues of capsazepine may yield compounds that could selectively inhibit HCN channels and prove useful for the treatment of neurological disorders where a role for HCN channels has been described.
Gill
Catherine H
CH
Neurology & GI CEDD, GlaxoSmithKline, New Frontiers Science Park North, Third Avenue, Harlow, Essex CM19 5AW.
Randall
Andrew
A
Bates
Stewart A
SA
Hill
Kerstin
K
Owen
Davina
D
Larkman
Phil M
PM
Cairns
William
W
Yusaf
Shahnaz P
SP
Murdock
Paul R
PR
Strijbos
Paul J L M
PJ
Powell
Andrew J
AJ
Benham
Christopher D
CD
Davies
Ceri H
CH
D000818:Animals; D001552:Benzazepines / Q000494:pharmacology; D016466:CHO Cells; D002211:Capsaicin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000494:pharmacology; D015221:Potassium Channels; D011743:Pyrimidines / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D001552:Benzazepines; D054815:Cyclic Nucleotide-Gated Cation Channels; C578665:HCN1 protein, human; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D015221:Potassium Channels; D011743:Pyrimidines; C082246:ICI D2788; D000242:Cyclic AMP; C071423:capsazepine; D011188:Potassium; D002211:Capsaicin; C043636:zatebradine