Isoform-specific regulation of mood behavior and pancreatic beta cell and cardiovascular function by L-type Ca 2+ channels.

Ca(v)1.2 and Ca(v)1.3 L-type Ca(2+) channels (LTCCs) are believed to underlie Ca(2+) currents in brain, pancreatic beta cells, and the cardiovascular system. In the CNS, neuronal LTCCs control excitation-transcription coupling and neuronal plasticity. However, the pharmacotherapeutic implications of CNS LTCC modulation are difficult to study because LTCC modulators cause cardiovascular (activators and blockers) and neurotoxic (activators) effects. We selectively eliminated high dihydropyridine (DHP) sensitivity from Ca(v)1.2 alpha 1 subunits (Ca(v)1.2DHP-/-) without affecting function and expression. This allowed separation of the DHP effects of Ca(v)1.2 from those of Ca(v)1.3 and other LTCCs. DHP effects on pancreatic beta cell LTCC currents, insulin secretion, cardiac inotropy, and arterial smooth muscle contractility were lost in Ca(v)1.2DHP-/- mice, which rules out a direct role of Ca(v)1.3 for these physiological processes. Using Ca(v)1.2DHP-/- mice, we established DHPs as mood-modifying agents: LTCC activator-induced neurotoxicity was abolished and disclosed a depression-like behavioral effect without affecting spontaneous locomotor activity. LTCC activator BayK 8644 (BayK) activated only a specific set of brain areas. In the ventral striatum, BayK-induced release of glutamate and 5-HT, but not dopamine and noradrenaline, was abolished. This animal model provides a useful tool to elucidate whether Ca(v)1.3-selective channel modulation represents a novel pharmacological approach to modify CNS function without major peripheral effects.

Cav1.4alpha1 subunits can form slowly inactivating dihydropyridine-sensitive L-type Ca2+ channels lacking Ca2+-dependent inactivation.

The neuronal L-type calcium channels (LTCCs) Cav1.2alpha1 and Cav1.3alpha1 are functionally distinct. Cav1.3alpha1 activates at lower voltages and inactivates more slowly than Cav1.2alpha1, making it suitable to support sustained L-type Ca2+ inward currents (ICa,L) and serve in pacemaker functions. We compared the biophysical and pharmacological properties of human retinal Cav1.4alpha1 using the whole-cell patch-clamp technique after heterologous expression in tsA-201 cells with other L-type alpha1 subunits. Cav1.4alpha1-mediated inward Ba2+ currents (IBa) required the coexpression of alpha2delta1 and beta3 or beta2a subunits and were detected in a lower proportion of transfected cells than Cav1.3alpha1. IBa activated at more negative voltages (5% activation threshold; -39mV; 15 mm Ba2+) than Cav1.2alpha1 and slightly more positive than Cav1.3alpha1. Voltage-dependent inactivation of IBa was slower than for Cav1.2alpha1 and Cav1.3alpha1( approximately 50% inactivation after 5 sec; alpha2delta1 + beta3 coexpression). Inactivation was not increased with Ca2+ as the charge carrier, indicating the absence of Ca2+-dependent inactivation. Cav1.4alpha1 exhibited voltage-dependent, G-protein-independent facilitation by strong depolarizing pulses. The dihydropyridine (DHP)-antagonist isradipine blocked Cav1.4alpha1 with approximately 15-fold lower sensitivity than Cav1.2alpha1 and in a voltage-dependent manner. Strong stimulation by the DHP BayK 8644 was found despite the substitution of an otherwise L-type channel-specific tyrosine residue in position 1414 (repeat IVS6) by a phenylalanine. Cav1.4alpha1 + alpha2delta1 + beta channel complexes can form LTCCs with intermediate DHP antagonist sensitivity lacking Ca2+-dependent inactivation. Their biophysical properties should enable them to contribute to sustained ICa,L at negative potentials, such as required for tonic neurotransmitter release in sensory cells and plateau potentials in spiking neurons.

Molecular characterization of rabbit phospholipid transfer protein: choroid plexus and ependyma synthesize high levels of phospholipid transfer protein.

Phospholipid transfer protein (PLTP) plays an important role in plasma lipoprotein metabolism. However, PLTP is expressed in a wide range of tissues suggesting additional local functions. To analyze the tissue distribution of PLTP in an animal with high-level expression of the structurally and functionally related CETP, we have cloned the full-length cDNA of rabbit PLTP (1,796 bp). Rabbit PLTP cDNA shows high homology to human, murine, and porcine PLTP cDNA, averaging 86.1%, 80.4%, and 86.1%, respectively. Interestingly, the C-terminus contains a unique seven amino acid insertion not found in previously characterized mammalian PLTPs. In clear contradistinction to human PLTP, rabbit PLTP mRNA was prominent in brain. In situ hybridization studies revealed specific, high-level synthesis of PLTP mRNA in choroid plexus and ependyma, the organs responsible for production of cerebrospinal fluid. Consistent with these findings, PLTP activity in cerebrospinal fluid amounted to 23% +/- 3% of that in rabbit plasma. In contrast, neither CETP mRNA nor CETP activity were detectable in rabbit brain.A role of PLTP in the central nervous system could involve some of its actions previously established in vitro, like proteolysis of apolipoproteins, and be physiologically relevant for neurodegenerative disorders such as Alzheimer's disease.

Functional consequences of P/Q-type Ca2+ channel Cav2.1 missense mutations associated with episodic ataxia type 2 and progressive ataxia.

We have investigated the functional consequences of three P/Q-type Ca(2+) channel alpha1A (Ca(v)2.1alpha(1)) subunit mutations associated with different forms of ataxia (episodic ataxia type 2 (EA-2), R1279Stop, AY1593/1594D; progressive ataxia (PA), G293R). Mutations were introduced into human alpha1A cDNA and heterologously expressed in Xenopus oocytes or tsA-201 cells (with alpha(2)delta and beta1a) for electrophysiological and biochemical analysis. G293R reduced current density in both expression systems without changing single channel conductance. R1279Stop and AY1593/1594D protein were expressed in tsA-201 cells but failed to yield inward barium currents (I(Ba)). However, AY1593/1594D mediated I(Ba) when expressed in oocytes. G293R and AY1593/1594D shifted the current-voltage relationship to more positive potentials and enhanced inactivation during depolarizing pulses (3 s) and pulse trains (100 ms, 1 Hz). Mutation AY1593/1594D also slowed recovery from inactivation. Single channel recordings revealed a change in fast channel gating for G293R evident as a decrease in the mean open time. Our data support the hypothesis that a pronounced loss of P/Q-type Ca(2+) channel function underlies the pathophysiology of EA-2 and PA. In contrast to other EA-2 mutations, AY1593/1594D and G293R form at least partially functional channels.