Abstract
Dihydropyridines (DHPs) block L-type Ca2+ channels more potently at depolarized membrane potentials, consistent with high affinity binding to the inactivated state. Nisoldipine (a DHP antagonist) blocks the smooth muscle channel more potently than the cardiac one, a phenomenon observed not only in native channels but also in expressed channels. We examined whether this tissue specificity was attributable to differences of inactivation in the two channel types. We expressed cardiac or smooth muscle α1C subunits in combination with β2a and α2/δ subunits in human embryonic kidney cells, and used 2 mm Ca2+ as the permeant ion. This system thus reproduces the in vivo topology and charge carrier of the channels while facilitating comparison of the two α1C splice variants. Both voltage-dependent and isoform-specific sensitivity of 10 nm nisoldipine inhibition of the channel were demonstrated, with the use of −100 mV as the holding potential for fully reprimed channels and −65 mV to populate the inactivated state. Under drug-free conditions, we characterized fast inactivation (1-sec prepulses) and slow inactivation (3 min prepulses) in the two isoforms. Inactivation parameters were not statistically different in the two channel isoforms; if anything, cardiac channels tended to inactivate more than the smooth muscle channels at relevant voltages. Likewise, the voltage-dependent activation was identical in the two isoforms. We thus conclude that the more potent nisoldipine inhibition of smooth muscle versus cardiac L-type Ca2+ channels is not attributable to differences in channel inactivation or activation. Intrinsic, gating-independent DHP receptor binding affinity differences must be invoked to explain the isoform-specific sensitivity of the DHP block.
Footnotes
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Send reprint requests to: Eduardo Marban, M.D., Ph.D., 844 Ross Bldg., Johns Hopkins University, School of Medicine, Baltimore, MD 21205. E-mail: marban{at}welchlink.welch.jhu.edu
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This work was supported by National Institutes of Health Grants R01-HL52768 (E.M.) and T32-HL07227 (H.H.).
- Abbreviations:
- DHP
- dihydropyridine
- α1C-a
- the α1 subunit of the cardiac L-type Ca2+ channel
- α1C-b
- the α1 subunit of the smooth muscle L-type Ca2+ channel
- ANCOVA
- analysis of covariance
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEK
- human embryonic kidney
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- ICa
- peak Ca2+ current through the L-type Ca2+channels
- SM
- smooth muscle
- Vh
- membrane holding potential
- Received December 8, 1997.
- Accepted February 4, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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