Voltage-gated Calcium Channel Function Research Articles

Scrapie-infected GT1-1 cells show impaired function of voltage-gated N-type calcium channels (Ca v 2.2) which is ameliorated by quinacrine treatment

Prions are transmissible pathogens that cause neurodegenerative diseases, although the mechanisms behind the nervous system dysfunctions are unclear. To study the effects of a prion infection on voltage-gated calcium channels, scrapie-infected gonadotropin-releasing hormone neuronal cells (ScGT1-1) in culture were depolarized by KCl and calcium responses recorded. Lower calcium responses were observed in infected compared to uninfected cells. This effect was still observed when L-type calcium channels were blocked by nimodipine. After inhibition of N-type calcium channels with ω-conotoxin GVIA, there was no difference in calcium responses. The calcium responses after nimodipine treatment became progressively lower during infection, but there was no major loss of the cellular prion protein (PrP C) or marked increase in accumulation of the abnormal prion protein (PrP Sc) in the cultures. These results indicate that scrapie infection causes a dysfunction of voltage-gated N-type calcium channels, which is exacerbated slowly over time. Quinacrine treatment cleared PrP Sc and restored calcium responses in the ScGT1-1 cultures.

Nitric Oxide/cGMP-Induced Inhibition of Calcium and Chloride Currents in Retinal Pericytes

In the CNS, contractile pericytes positioned on endothelium-lined lumens appear to play a role in regulating capillary blood flow. This function may be particularly important in the retina where pericytes are more numerous than in other tissues. Despite the importance of pericytes, knowledge of the effects of vasoactive molecules, such as nitric oxide (NO), on the physiology of these cells is limited. Since it is likely that ion channels play a role in the response of pericytes to signaling molecules from other cells, we used the perforated-patch configuration of the patch-clamp technique to record the whole-cell currents of pericytes located on microvessels freshly isolated from the rat retina. We found that voltage-gated calcium currents and calcium-activated chloride currents were inhibited during exposure to the NO donor, sodium nitroprusside (SNP). 8-Bromo-cyclic guanosine monophosphate (cGMP) mimicked these effects. In contrast, neither SNP nor the cGMP analog significantly affected the potassium or nonspecific cation conductances, which establish the resting membrane potential of retinal pericytes. Consistent with endogenous NO suppressing pericyte channel activity, exposure of isolated microvessels to an inhibitor of NO synthase increased the calcium and chloride currents. Since our experiments indicate that chloride channel activity is dependent, in part, upon the function of voltage-gated calcium channels, we postulate that a NO/cGMP-mediated inhibition of calcium channels reduces calcium influx and, thereby, lessens the opening of the calcium-activated chloride channels. This may be one mechanism by which NO decreases the contractile tone of pericytes.

T-type calcium channels facilitate insulin secretion by enhancing general excitability in the insulin-secreting beta-cell line, INS-1.

The present study addresses the function of T-type voltage-gated calcium channels in insulin-secreting cells. We used whole-cell voltage and current recordings, capacitance measurements, and RIA techniques to determine the contribution of T-type calcium channels in modulation of electrical activity and in stimulus-secretion coupling in a rat insulin secreting cell line, INS-1. By employing a double pulse protocol in the current-clamp mode, we found that activation of T-type calcium channels provided a low threshold depolarizing potential that decreased the latency of onset of action potentials and furthermore increased the frequency of action potentials, both of which are abolished by administration of nickel chloride (NiCl2), a selective T-type calcium channel blocker. Moreover application of high frequency stimulation, as compared with low frequency stimulation, caused a greater change in membrane capacitance (deltaCm), suggesting higher insulin secretion. We demonstrated that glucose stimulated insulin secretion in INS-1 is reduced dose dependently by NiCl2. We conclude that T-type calcium channels facilitate insulin secretion by enhancing the general excitability of these cells. In light of the pathological effects of both hypo and hyperinsulinemia, the T-type calcium channel may be a therapeutic target.

Metabotropic glutamate receptor modulation of synaptic transmission in corticostriatal co-cultures: Role of calcium influx

Modulation of excitatory glutamatergic transmission at corticostriatal synapses by a metabotropic glutamate receptor (mGluR) was examined using a newly developed cell culture preparation in which small explants of cortical tissue are grown in co-culture with isolated striatal neurons. Electrical stimulation of cortical tissue evoked excitatory postsynaptic currents (eEPSCs) observed during tight-seal, whole-cell recordings from striatal neurons. Transmission was mediated by activation of AMPA/kainate-type glutamate receptors. The mGluR agonists, 1 SR,3 RS-ACPD and DCG-IV, reduced eEPSC amplitude. The effect of 1 SR,3 RS-ACPD increased in a concentration-dependent manner. Application of phorbol diacetate (PDAc) potentiated eEPSC amplitude and reduced the inhibitory effect of mGluR activation. Pretreatment with pertussis toxin (PTX) also reduced inhibition by 1 SR,3 RS-ACPD. Under conditions in which transmission was independent of the function of voltage-gated calcium channels, mGluR activation reduced the frequency of occurrence of miniature EPSCs (mEPSCs), but did not alter mEPSC amplitude. This effect of mGluR activation was reduced by PDAc treatment. mGluR activation modulates glutamatergic transmission via a presynaptic autoreceptor at corticostriatal synapses in this newly-developed corticostriatal co-culture preparation as in striatal slices. Modulation of transmission occurs whether or not transmission involves activation of voltage-gated calcium channels. Furthermore, many of the characteristics of mGluR modulation of eEPSCs are shared by mGluR modulation of mEPSCs. These findings indicate that mechanisms downstream from calcium entry may contribute to modulation of synaptic transmission by mGluR autoreceptors.

Altered muscle calcium channel binding kinetics in autoimmune motoneuron disease.

While skeletal muscle is not apparently affected directly in amyotrophic lateral sclerosis (ALS), immunoglobulin G fractions purified from patients with ALS (ALS IgG) bind dihydropyridine (DHP)-sensitive L-type voltage-gated calcium channel (VGCC) antigen isolated from skeletal muscle in ELISA and Western immunoblot, and alter VGCC function in vitro. To determine whether muscle VGCC properties are altered in ALS, VGCC-enriched subsarcolemmal membrane fractions were prepared from biopsied quadriceps muscle of patients with ALS, with other neurologic diseases, or without apparent muscle disease, and tested for DHP binding with [3H]PN200-110. ALS muscle VGCCs possessed eightfold higher binding affinities for [3H]PN200-110 than did VGCCs from muscle fractions of most other patients, independent of denervation-induced increases in DHP binding site number. Similarly elevated DHP binding affinities were observed in specimens from patients with autoimmune motor neuropathies, suggesting that ALS and immune mediated motoneuron disease share skeletal muscle L-type VGCC alterations.

Amyotrophic lateral sclerosis immunoglobulins increase Ca2+ currents in a motoneuron cell line

The sporadic form of amyotrophic lateral sclerosis (ALS) is an idiopathic and eventually lethal disorder causing progressive degeneration of cortical and spinal motoneurons. Recent studies have shown that the majority of patients with sporadic ALS have serum antibodies that bind to purified L-type voltage-gated calcium channels and that antibody titer correlates with the rate of disease progression. Furthermore, antibodies purified from ALS patient sera have been found to alter the physiologic function of voltage-gated calcium channels in nonmotoneuron cell types. Using whole-cell patch-clamp techniques, immunoglobulins purified from sera of 5 of 6 patients with sporadic ALS are now shown to increase calcium currents in a hybrid motoneuron cell line, VSC4.1. These calcium currents are blocked by the polyamine funnel-web spider toxin FTX, which has previously been shown to block Ca2+ currents and evoked transmitter release at mammalian motoneuron terminals. These data provide additional evidence linking ALS to an autoimmune process and suggest that antibody-induced increases in calcium entry through voltage-gated calcium channels may occur in motoneurons in this disease, with possible deleterious effects in susceptible neurons.

Amyotrophic lateral sclerosis patient antibodies label Ca2+ channel alpha 1 subunit.

Sporadic amyotrophic lateral sclerosis is an idiopathic human degenerative disease of spinal cord and brain motor neurons. Prior studies demonstrated that most patients with amyotrophic lateral sclerosis possess immunoglobulins that bind to purified L-type voltage-gated calcium channels, that titers of anti-voltage-gated calcium channel antibodies correlate with disease progression rates, and that amyotrophic lateral sclerosis patient-derived antibodies (ALS IgG) produce electrophysiological changes in the function of voltage-gated calcium channels. Using Western transfer immunoblots and enzyme-linked immunosorbent assays, the calcium ionophore-forming alpha 1 subunit of the voltage-gated calcium channel is now identified as the major voltage-gated calcium channel antigen to which ALS IgG binds. Additionally, the binding of an L-type voltage-gated calcium channel alpha 1 subunit-directed monoclonal antibody, which itself mimics the effects of ALS IgG on skeletal muscle voltage-gated calcium channel currents, is selectively prevented by preaddition of ALS IgG. Voltage-gated calcium channel-binding IgG from patients with Lambert-Eaton myasthenic syndrome appears to be differentiated from ALS IgG by the reactivity of the former to both alpha 1 and beta subunits of the calcium channel. These assays provide further evidence linking amyotrophic lateral sclerosis to an autoimmune process, and suggest one means to differentiate immunoglobulins from patients with amyotrophic lateral sclerosis from those of patients with another autoimmune disease expressing calcium channel antibodies.

Calcium channel blockade attenuates angiotensin II-Induced drinking in rats

Lateral ventricular administration of angiotensin II (ANGII) produces potent dipsogenic effects in water-sated rats. ANG II seems to require functional voltage-gated calcium channels on neurons throughout circumventricular brain sites to exert its effects. Although there are at least three types of calcium channels, only L-type calcium channel-blocking drugs have been reported to decrease drinking. (4-(4-Benzofurazanyl)-l-4-dihydro-2,6-dimethyl-3,5-pyridine-dicarboxylic acid methyl 1-methyl-ethyl ester) [PN 200-110; isradipine (ISR)], a selective L-type calcium channel blocker, has been shown to attenuate significantly the intake of sweetened water in water-sated rats following either peripheral or ICV administration, but ISR does not affect plain-water intake in water-deprived rats. The present experiment was designed to determine whether ISR would attenuate ANG II-induced drinking that is not either motivated by palatability or dependent on deprivation. Rats, each fitted with chronic indwelling ventricular cannulae, were pretreated with ISR (0.3, 3.0, and 30 μg/rat; ICV). ANG II (40 ng/rat; ICV) was administered 10 min later and rats were allowed free access to water for 15 min. Injections of ANG II plus saline and ANG II plus the ISR vehicle (dimethyl sulfoxide) did not attenuate ANG II-induced polydipsia, whereas ANG II+ISR (0.3 and 3.0 μg) attenuated ANG II-induced drinking to 62 and 22% of control, respectively. Results with the 30-μg dose were not different from the 3.0 dose. In conclusion, our findings support the hypothesis that ANG II-induced polydipsia is mediated, at least in part, by calcium voltage-dependent channels as the pretreatment with ISR effectively blocked ANG II-induced drinking.

Structure and function of voltage-gated sodium and calcium channels

The primary structures of the Na+ channel a-subunits from several species have now been deduced from cDNA sequences, and complete primary structures of all of the subunits of skeletal muscle L-type Ca 2+ channels have been defined. Current research on voltage-gated ion channels is focusing on defining the structural components responsible for specific aspects of channel function. Recent experiments have identified regions of these channels that are important for voltage-dependent activation and inactivation, ion conductance, regulation by protein phosphorylation, and modulation by drugs and neurotoxins using a combination of antibody mapping and site-directed mutagenesis approaches. The results form the outlines of a structural map of ion channel function.

Inactivation of calcium channels

Rapid progress in our understanding of the properties and functions of voltage-gated calcium channels has produced the need for an update to our previous review of calcium inactivation. The major elements of change included in this review are: 1. 1. The existence of multiple forms of voltage-sensitive Ca + channels, with distinctive single channel properties, thus necessitating a reappraisal of properties deduced from macroscopic current recordings, particularly of the processes of activation and inactivation. 2. 2. The differences in biochemical properties between channel types are reflected in their differences in divalent selectivity, their requirement for metabolic maintenance and their mechanism of inactivation. These properties appear to divide the channels into two categories which may relate to their molecular structures. Further subgroupings, based upon the voltage thresholds, have also been observed. 3. 3. Molecular properties of one class of channels have been elucidated, which correlate with the observed biochemistry of channel modulation and inactivation. 4. 4. An enzymatic process underlying the mechanism of Ca 2+-dependent inactivation has been elucidated and may serve as a model for other modulatory systems. The interweaving of the properties of these Ca 2+ channels, with their spatial distributions and their influence upon other channel types, acts to transduce and integrate information within cells.