Mibefradil is a Ca2+ channel antagonist that inhibits both T-type and high-voltage-activated Ca2+ channels. We previously showed that block of high-voltage-activated channels by mibefradil occurs through the production of an active metabolite by intracellular hydrolysis. In the present study, we modified the structure of mibefradil to develop a nonhydrolyzable analog, (1S, 2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride (NNC 55-0396), that exerts a selective inhibitory effect on T-type channels. The acute IC(50) of NNC 55-0396 to block recombinant alpha(1)G T-type channels in human embryonic kidney 293 cells was approximately 7 microM, whereas 100 microM NNC 55-0396 had no detectable effect on high-voltage-activated channels in INS-1 cells. NNC 55-0396 did not affect the voltage-dependent activation of T-type Ca2+ currents but changed the slope of the steady-state inactivation curve. Block of T-type Ca2+ current was partially relieved by membrane hyperpolarization and enhanced at a high-stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca2+ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca2+ currents, arguing against a cytoplasmic route of action. After incubating cells from an insulin-secreting cell line (INS-1) with NNC 55-0396 for 20 min, mass spectrometry did not detect the mibefradil metabolite that causes L-type Ca2+ channel inhibition. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L-type Ca2+ channels, thus rendering it more selective to T-type Ca2+ channels.
"To analyze the role played by LT-VACCs in synaptic integration, it is necessary to count on specific blockers devoid of any effect on the high-threshold VACCs that participate in synaptic transmission. The effects of three pharmacological agents known as T-type Ca 2ϩ channel blockers were studied: amiloride (Huguenard and Prince 1992; Tang et al. 1988), Ni 2ϩ (Huguenard and Prince 1992; Lee et al. 1999; Regan 1991), and NNC, a structural analog of mibefradil (Huang et al. 2004; Johnston and Delaney 2010). "
"However, mibefradil also blocks high-VACCs (Fang and Osterrieder, 1991; Bezprozvanny and Tsien, 1995; Viana et al., 1997). Later on, mibefradil derivative NNC 55-0396 (NNC) emerged as a more selective T-type channel blocker (Huang et al., 2004; Li et al., 2005). Unfortunately, a selective T channel blocker with potential scarce side effects is still an unmet therapeutic goal (Arranz-Tagarro et al., 2014). "
"In this case, the sag response was absent indicating that Ih may not contribute to PIR. The possible contribution of T-type channels to the rebound response was also investigated using NCC55-0396, a mibefradil derivative that has been reported to block T-type channels . The amplitude of the rebound depolarization was decreased to ∼59±10% of the control mean value by NCC55-0396 application (n = 7), as illustrated in Figure 3B for a representative motoneuron antidromically identified. "
[Show abstract][Hide abstract] ABSTRACT: Voltage-gated Ca2+ (CaV) channels are transmembrane proteins comprising three subfamilies named CaV1, CaV2 and CaV3. The CaV3 channel subfamily groups the low-voltage activated Ca2+ channels (LVA or T-type) a significant role in regulating neuronal excitability. CaV3 channel activity may lead to the generation of complex patterns of action potential firing such as the postinhibitory rebound (PIR). In the adult spinal cord, these channels have been found in dorsal horn interneurons where they control physiological events near the resting potential and participate in determining excitability. In motoneurons, CaV3 channels have been found during development, but their functional expression has not yet been reported in adult animals. Here, we show evidence for the presence of CaV3 channel-mediated PIR in motoneurons of the adult turtle spinal cord. Our results indicate that Ni2+ and NNC55-0396, two antagonists of CaV3 channel activity, inhibited PIR in the adult turtle spinal cord. Molecular biology and biochemical assays revealed the expression of the CaV3.1 channel isotype and its localization in motoneurons. Together, these results provide evidence for the expression of CaV3.1 channels in the spinal cord of adult animals and show also that these channels may contribute to determine the excitability of motoneurons.
PLoS ONE 09/2014; 9(9):e108187. DOI:10.1371/journal.pone.0108187 · 3.23 Impact Factor
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