A member of a new subclass of the voltage‐activated sodium channel genes has been cloned from the human medullary thyroid carcinoma (hMTC) cell line. The cDNA of hNE‐Na (human neuroendocrine sodium ...channel) encodes a 1977 amino acid protein which phylogenetically represents a link between sodium channels isolated from skeletal muscle and brain. The hNE‐Na alpha subunit was transiently expressed in human embryonic kidney cells either alone or in combination with the human sodium channel beta 1 subunit. The channel exhibited rapid activation and inactivation kinetics, and was blocked by tetrodotoxin and cadmium with IC50 values of 24.5 nM and 1.1 mM, respectively. Action potentials were generated in cells expressing high levels of hNE‐Na. Northern blot and reverse transcription‐polymerase chain reaction (RT‐PCR) analyses demonstrated its expression in hMTC cells, in a C‐cell carcinoma, and in thyroid and adrenal gland. Transcripts were not identified in pituitary gland, brain, heart, liver or kidney, indicating that the hNE‐Na is a sodium channel solely expressed in neuroendocrine cells.
The pharmacological properties of the expressed murine T-type alpha(1G) channel were characterized using the whole cell patch clamp configuration. Ba(2+) or Ca(2+) were used as charge carriers. Both ...I(Ba) and I(Ca) were blocked by Ni(2+) and Cd(2+) with IC(50) values of 0.47+/-0.04 and 1.13+/-0.06 mM (Ni(2+)) and 162+/-13 and 658+/-23 microM (Cd(2+)), respectively. Ni(2+), but not Cd(2+), modified the gating of channel activation. Ni(2+) consistently accelerated channel deactivation while Cd(2+) had a similar effect only on I(Ca). The alpha(1G) channel was potently blocked by mibefradil in a dose- and voltage-dependent manner. I(Ba) was moderately blocked by phenytoin (IC(50) 73.9+/-1.9 microM) and was resistant to the block by valproate. Also 3 mM ethosuximide blocked 20 and 35% of the I(Ba) at a HP of -100 and -60 mV, respectively, while 5 mM amiloride inhibited I(Ba) by 38% and significantly slowed current activation. The alpha(1G) channel was not affected by 10 microM tetrodotoxin. Both 1 microM (+)isradipine and 10 microM nifedipine inhibited 18 and 14% of I(Ba) amplitude at a HP of -100 mV, and 23% and 29% of I(Ba) amplitude at a HP of -60 mV, respectively. The alpha(1G) current was minimally activated by 1 microM Bay K 8644.
Intramembrane charge movement originating from Cav3.1 (T‐type) channel expressed in HEK 293 cells was investigated. Ion current was blocked by 1 mM La3+. Charge movement was detectable for ...depolarizations above ∼−70 mV and saturated above +60 mV. The voltage dependence of charge movement followed a single Boltzmann function with half‐maximal activation voltage +12.9 mV and +12.3 mV and with slopes of 22.4 mV and 18.1 mV for the ON‐ and OFF‐charge movement, respectively. Inactivation of ICa by prolonged depolarization pulse did not immobilize intramembrane charge movement in the Cav3.1 channel.
Paroxysmal extreme pain disorder (PEPD), previously known as familial rectal pain (FRP, or OMIM 167400), is an inherited condition characterized by paroxysms of rectal, ocular, or submandibular pain ...with flushing. A genome-wide linkage search followed by mutational analysis of the candidate gene
SCN9A, which encodes hNa
v1.7, identified eight missense mutations in 11 families and 2 sporadic cases. Functional analysis in vitro of three of these mutant Na
v1.7 channels revealed a reduction in fast inactivation, leading to persistent sodium current. Other mutations in
SCN9A associated with more negative activation thresholds are known to cause primary erythermalgia (PE). Carbamazepine, a drug that is effective in PEPD, but not PE, showed selective block of persistent current associated with PEPD mutants, but did not affect the negative activation threshold of a PE mutant. PEPD and PE are allelic variants with distinct underlying biophysical mechanisms and represent a separate class of peripheral neuronal sodium channelopathy.
A member of the low-voltage-activated calcium channel family was identified in mouse brain by taking advantage of amino acid sequences that have been evolutionary conserved. The identified sequence ...is similar to that of the recently cloned rat alpha1G T-type calcium channel, but there are differences in two insertions in the intracellular connecting loops. Northern blot analysis indicates that its expression is strong in the brain. In situ hybridization revealed that, in mouse brain, the alpha1G mRNA is found in the cerebellum, hippocampus, thalamus and olfactory bulb. In contrast to L-type calcium channel currents, IBa and ICa through the alpha1G channel expressed in HEK293 cells did not differ in terms of current density, voltage dependence of current activation, inactivation and deactivation, and speed of recovery from voltage-dependent inactivation. The kinetics of ICa inactivation were significantly slower than those of IBa. The expressed alpha1G channel has a relatively high sensitivity to mibefradil, but is only slightly affected by Ni2+.
The current through the L‐type calcium channel is inhibited and stimulated by distinct dihydropyridines at very low concentrations. The molecular determinants for the high affinity block and ...stimulation were investigated using chimeras between the class C and E calcium channels. Mutation of three amino acids in the last putative transmembrane segment (IVS6) of the alpha1C subunit decreased the affinity for (+)isradipine 100‐fold without significantly affecting the basic properties of the expressed channel. Mutation of two of these three amino acids completely abolished the stimulatory effect of the calcium channel agonist Bay K 8644. These mutations only slightly affected the blocking efficacy of mibefradil and the phenylalkylamine devapamil. Three distinct but adjacently located amino acids mediated the high affinity block by devapamil. These results suggest that the IVS6 segment of the alpha1C subunit is critical for the high affinity interaction between the L‐type calcium channel and the calcium channel agonist Bay K 8644 and the two antagonists isradipine and devapamil.
High-voltage activated calcium channels are modulated by a series of auxiliary proteins, including those of the alpha(2)delta family. Until recently, only a single alpha(2)delta subunit was known, ...but two further members, alpha(2)delta-2 and -3, have since been identified. In this study, the structure of these two novel subunits has been characterized and binding of the antiepileptic drug gabapentin investigated. Using antibodies directed against the amino terminal portion of the proteins, the gross structure of the subunits could be analyzed by Western blotting. Similar to alpha(2)delta-1, both alpha(2)delta-2 and -3 subunits consist of two proteins-a larger alpha(2) and a smaller delta that can be separated by reduction. The subunits are also highly N-glycosylated with approximately 30 kDa of their mass consisting of oligosaccharides. alpha(2)delta-1 was detected in all mouse tissues studied, whereas alpha(2)delta-2 was found at high levels in brain and heart. The alpha(2)delta-3 subunit was observed only in brain. alpha(2)delta-1 and alpha(2)delta-2, but not alpha(2)delta-3, were found to bind gabapentin. The K(d) value of gabapentin binding to alpha(2)delta-2 was 153 nM compared with the higher affinity binding to alpha(2)delta-1 (K(d) = 59 nM).
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