Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+-induced glucagon exocytosis in pancreas

Hormones such as glucagon are secreted by Ca²⁺-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic β-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca²⁺-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca²⁺ sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca²⁺ sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca²⁺-triggered glucagon release from α-cells than insulin secretion from β-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca²⁺-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca²⁺-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic α-cells lacking synaptotagmin-7 exhibited little Ca²⁺-induced exocytosis, whereas all other physiological and morphological parameters of the α-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca²⁺ sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca²⁺ sensors in neurotransmitter, neuropeptide, and hormone secretion.     

Amplification of exocytosis by Ca2+-induced Ca2+ release in INS-1 pancreatic β cells

Modulation of spontaneous electrical activities (slow waves, pacemaker potentials and follower potentials) in response to hyperpolarization produced by the ATP-sensitive K⁺ channel openers (KCOs) pinacidil or nicorandil was investigated in smooth muscle tissues of the guinea-pig stomach antrum. With hyperpolarization, the amplitude of slow waves and follower potentials was reduced and that of pacemaker potentials was increased, with a minor modulation of their frequency. The attenuation of slow waves was associated with an inhibition of the 1st component and abolition of the 2nd component. All these actions of KCOs were antagonized by glibenclamide. An increase in the extracellular K⁺ concentration prevented the KCO-induced hyperpolarization with partial restoration of slow waves, suggesting that the inhibition was produced mainly by a decrease in membrane resistance. Exposure of tissues to KCOs for a long period of time (> 20 min) resulted in the reappearance of slow waves displaying both 1st and 2nd components. The 2nd component of the slow wave, which displayed a slower recovery, was inhibited again by 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-sensitive K⁺ channels. Noradrenaline hyperpolarized the membrane by activating apamin-sensitive K⁺ channels and increased the amplitude and frequency of slow waves through activation of α₁-adrenoceptors, actions different from those of KCOs. Thus, inhibition of slow waves by KCOs may be primarily related to the decrease in amplitude of a passive electrotonic component, possibly due to a reduction of the input resistance. The hyperpolarization shifted the threshold potential for generation of the 2nd component of slow waves to negative levels, presumably due to modulation of mitochondrial functions.     

pH-dependent and -independent effects inhibit Ca2+-induced Ca2+ release during metabolic blockade in rat ventricular myocytes

We have investigated the role of changes of intracellular pH (pH_i) in the effects of metabolic blockade (cyanide plus 2-deoxyglucose) on Ca²⁺ release from the sarcoplasmic reticulum (SR) in rat ventricular myocytes. pH_i and cell length were measured simultaneously. Metabolic blockade decreased the frequency of Ca²⁺ waves, an effect previously shown to be due to inhibition of Ca²⁺ release from the SR. This was accompanied by an intracellular acidification. Intracellular acidification was produced in the absence of metabolic inhibition by application of sodium butyrate. A maintained intracellular acidosis produced a decrease of wave frequency. A hysteresis between pH_i and wave frequency was observed such that during the onset of the acidification the wave frequency decreased more than in the steady state. Comparison of the steady state relationship between pH_i and wave frequency showed that the decrease of wave frequency produced by metabolic blockade was greater than could be accounted for simply by the accompanying decrease of pH_i. In other experiments the buffering power of the solution was increased. Under these conditions, metabolic blockade produced no change of pH_i but the decrease of wave frequency persisted. We conclude that, although intracellular acidification occurs during metabolic blockade, it is not responsible for most of the inhibition of Ca²⁺ release from the SR.     

Modulation of Ca2+ release and Ca2+ oscillations in HeLa cells and fibroblasts by mitochondrial Ca2+ uniporter stimulation

The recent availability of activators of the mitochondrial Ca²⁺ uniporter allows direct testing of the influence of mitochondrial Ca²⁺ uptake on the overall Ca²⁺ homeostasis of the cell. We show here that activation of mitochondrial Ca²⁺ uptake by 4,4',4"-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or kaempferol stimulates histamine-induced Ca²⁺ release from the endoplasmic reticulum (ER) and that this effect is enhanced if the mitochondrial Na⁺–Ca²⁺ exchanger is simultaneously inhibited with CGP37157. This suggests that both Ca²⁺ uptake and release from mitochondria control the ability of local Ca²⁺ microdomains to produce feedback inhibition of inositol 1,4,5-trisphosphate receptors (InsP₃Rs). In addition, the ability of mitochondria to control Ca²⁺ release from the ER allows them to modulate cytosolic Ca²⁺ oscillations. In histamine stimulated HeLa cells and human fibroblasts, both PPT and kaempferol initially stimulated and later inhibited oscillations, although kaempferol usually induced a more prolonged period of stimulation. Both compounds were also able to induce the generation of Ca²⁺ oscillations in previously silent fibroblasts. Our data suggest that cytosolic Ca²⁺ oscillations are exquisitely sensitive to the rates of mitochondrial Ca²⁺ uptake and release, which precisely control the size of the local Ca²⁺ microdomains around InsP₃Rs and thus the ability to produce feedback activation or inhibition of Ca²⁺ release.     

Elementary properties of CaV1.3 Ca2+ channels expressed in mouse cochlear inner hair cells

Mammalian cochlear inner hair cells (IHCs) are specialized to process developmental signals during immature stages and sound stimuli in adult animals. These signals are conveyed onto auditory afferent nerve fibres. Neurotransmitter release at IHC ribbon synapses is controlled by L-type Ca_V1.3 Ca²⁺ channels, the biophysics of which are still unknown in native mammalian cells. We have investigated the localization and elementary properties of Ca²⁺ channels in immature mouse IHCs under near-physiological recording conditions. Ca_V1.3 Ca²⁺ channels at the cell pre-synaptic site co-localize with about half of the total number of ribbons present in immature IHCs. These channels activated at about −70 mV, showed a relatively short first latency and weak inactivation, which would allow IHCs to generate and accurately encode spontaneous Ca²⁺ action potential activity characteristic of these immature cells. The Ca_V1.3 Ca²⁺ channels showed a very low open probability (about 0.15 at −20 mV: near the peak of an action potential). Comparison of elementary and macroscopic Ca²⁺ currents indicated that very few Ca²⁺ channels are associated with each docked vesicle at IHC ribbon synapses. Finally, we found that the open probability of Ca²⁺ channels, but not their opening time, was voltage dependent. This finding provides a possible correlation between presynaptic Ca²⁺ channel properties and the characteristic frequency/amplitude of EPSCs in auditory afferent fibres.     

Calmodulin kinase modulates Ca2+ release in mouse skeletal muscle

Activation of the contractile machinery in skeletal muscle is initiated by the action-potential-induced release of Ca²⁺ from the sarcoplasmic reticulum (SR). Several proteins involved in SR Ca²⁺ release are affected by calmodulin kinase II (CaMKII)-induced phosphorylation in vitro , but the effect in the intact cell remains uncertain and is the focus of the present study. CaMKII inhibitory peptide or inactive control peptide was injected into single isolated fast-twitch fibres of mouse flexor digitorum brevis muscles, and the effect on free myoplasmic [Ca²⁺] ([Ca²⁺]_i) and force during different patterns of stimulation was measured. Injection of the inactive control peptide had no effect on any of the parameters measured. Conversely, injection of CaMKII inhibitory peptide decreased tetanic [Ca²⁺]_i by ≈25 %, but had no significant effect on the rate of SR Ca²⁺ uptake or the force-[Ca²⁺]_i relationship. Repeated tetanic stimulation resulted in increased tetanic [Ca²⁺]_i, and this increase was smaller after CaMKII inhibition. In conclusion, CaMKII-induced phosphorylation facilitates SR Ca²⁺ release in the basal state and during repeated contractions, providing a positive feedback between [Ca²⁺]_i and SR Ca²⁺ release.