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We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca2+ channel alpha1 subunit, alpha1H, from a human medullary thyroid carcinoma cell line. The alpha1H subunit is structurally similar to previously described alpha1 subunits. Northern blot analysis indicates that alpha1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba2+ currents recorded from human embryonic kidney 293 cells transiently expressing alpha1H activated at relatively hyperpolarized potentials (-50 mV), rapidly inactivated (tau = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing alpha1H. Single-channel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of approximately 9 pS. These channels are blocked by Ni2+ (IC50 = 6.6 microM) and the T-type channel antagonists mibefradil (approximately 50% block at 1 microM) and amiloride (IC50 = 167 microM). Thus, alpha1H-containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca2+ channels.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder in which antibodies to presynaptic calcium channels at the neuromuscular junction lead to muscle weakness. Around 60% of patients with LEMS have an associated small cell lung carcinoma (SCLC), which is known to express voltage-gated calcium channels (VGCCs). Immunoglobulin (IgG) from patients with LEMS causes inhibition of calcium flux in an SCLC cell line.1 This inhibition has been shown to correlate with disease severity.1 Neuronal VGCCs can be classified into P-, Q-, N-, L-, and R-type according to their electrophysiological and pharmacological properties. Around 92% of LEMS patients have antibodies that immunoprecipitate P/Q-type (125I-CTx-MVIIC labeled) VGCCs and around 30% have antibodies to N-type (125I-CTx-GVIA labeled) VGCCs.2 In addition, antibodies from LEMS patients have been shown to abolish the component of neurotransmitter release subserved by both P-type and Q-type VGCCs in sympathetic and parasympathetic neurons of mice injected with LEMS IgG.3 Neuronal VGCCs consist of an 1 subunit with and 2 subunits. Several of the human genes encoding the different types of VGCC have now been cloned and sequenced, including the 1A, 1B, 1D, 1E, 1–4, and 2 subunits. Human embryonic

Bovine adrenal chromaffin cells (BCC) were used to compare histamine- and angiotensin II-induced changes of inositol mono-, bis-, and trisphosphate (InsP1, InsP2, and InsP3, respectively) isomers, intracellular free Ca2+ ([Ca2+]i), and the pathways of inositol phosphate metabolism. Both agonists elevated [Ca2+]i by 200 nM 3-4 s after addition, but afterwards the histamine response was much more prolonged. Histamine and angiotensin II also produced similar four- to fivefold increases of Ins(1,4,5)P3 that peaked within 5 s. Over the first minute of stimulation, however, Ins(1,4,5)P3 formation was monophasic after angiotensin II, but biphasic after histamine, evidence supporting differential regulation of angiotensin II- and histamine-stimulated signal transduction. The metabolism of Ins(1,4,5)P3 by BCC homogenates was found to proceed via (a) sequential dephosphorylation to Ins(1,4)P2 and Ins(4)P, and (b) phosphorylation to inositol 1,3,4,5-tetrakisphosphate, followed by dephosphorylation to Ins(1,3,4)P3, Ins(1,3)P2, and Ins(3,4)P2, and finally to Ins(1 or 3)P. In whole cells, Ins(1 or 3)P only increased after histamine treatment. Additionally, Ins(1,3)P2 was the only other InsP2 besides Ins(1,4)P2 to accumulate within 1 min of agonist treatment [Ins(3,4)P2 did not increase]. These results support a correlation between the time course of Ins(1,4,5)P3 formation and the time course of [Ca2+]i transients and illustrate that Ca2(+)-mobilizing agonists can produce distinguishable patterns of inositol phosphate formation and [Ca2+]i changes in BCC. Different patterns of second-messenger formation are likely to be important in signal recognition and may encode agonist-specific information.

The effects of dibutyryl cyclic AMP (dbcAMP) and dibutyryl cyclic GMP (db-cGMP) were tested on Ca2+-ATPase, Mg2+-ATPase, and (Ca2++ Mg2+)-ATPase activities in lysed synaptosomes prepared from whole rat brains (minus cerebellum). At concentrations from 0.1 to 2.0 mM, db-cGMP produced a selective, concentration-dependent increase in Ca2+-ATPase activity. Both db-cGMP and db-cAMP slightly reduced Mg2+-ATPase activity, whereas neither compound had concentration-dependent effects on (Ca2++ Mg2+)-ATPase activity. These findings suggest that the Mg2+-independent, Ca2+-ATPase activity in rat brain is regulated by a cyclic GMP-dependent process. Further, the data provide evidence that the Ca2+-ATPase activity in lysed synaptosomal membranes represents an enzyme that is distinguishable from both the Mg2+- and (Ca2++ Mg2+-ATPase.