Contraction augments L-type Ca(2+) currents in adherent guinea-pig cardiomyocytes

As integrins are thought to function as mechanoreceptors, we studied whether they could mediate mechanical modulation of the L-type Ca(2+) channel current (I(Ca)) in guinea-pig cardiac ventricular myocytes (CVMs). CVMs were voltage clamped with 280 ms pulses from −45 to 0 mV at 0.5 Hz (1.8 mm[Ca(2+)](o), 22°C). Five minutes after whole-cell access (designated as 0 min) peak I(Ca) was determined from a current–voltage (I–V) curve. Additional recordings were made after 5, 10 and 15 min. At control, I(Ca) was not stable, but ran down during these periods. This run-down of I(Ca) was attenuated by soluble fibronectin (FN) and was changed to an enhancement of I(Ca) when CVMs were attached to FN-coated coverslips. Soluble peptide containing the integrin binding sequence of FN, Arg-Gly-Asp (RGD motif), did not modulate I(Ca); however, I(Ca) increased in stimulated CVMs attached to RGD peptide-coated coverslips. The effect was not specific to integrins, because attachment to poly-d-lysine-coated coverslips also augmented I(Ca) in stimulated CVMs. Augmentation of I(Ca) by immobilized FN required rhythmical contraction of attached CVMs, because it was attenuated without electrical stimulation and after cell dialysis with the calcium chelator BAPTA. Furthermore, contraction-induced augmentation of I(Ca) in FN-attached CVMs was sensitive to inhibition of protein kinase C (PKC; by Ro-31–8220), inhibition of tyrosine kinase activity (herbimycin A) and cytoskeletal depolymerization (cytochalasin D or colchicine). We attribute augmentation of I(Ca) to the activation of signalling cascades by shear forces that are generated when CVMs contract against attachment; in vivo similar signals may occur when CVMs contract against attachment of integrins to the extracellular matrix

Retinoschisin, a New Binding Partner for L-type Voltage-gated Calcium Channels in the Retina*

The L-type voltage-gated calcium channels (L-VGCCs) are activated under high depolarization voltages. They are vital for diverse biological events, including cell excitability, differentiation, and synaptic transmission. In retinal photoreceptors, L-VGCCs are responsible for neurotransmitter release and are under circadian influences. However, the mechanism of L-VGCC regulation in photoreceptors is not fully understood. Here, we show that retinoschisin, a highly conserved extracellular protein, interacts with the L-VGCCα1D subunit and regulates its activities in a circadian manner. Mutations in the gene encoding retinoschisin (RS1) cause retinal disorganization that leads to early onset of macular degeneration. Since ion channel activities can be modulated through interactions with extracellular proteins, disruption of these interactions can alter physiology and be the root cause of disease states. Co-immunoprecipitation and mammalian two-hybrid assays showed that retinoschisin and the N-terminal fragment of the L-VGCCα1 subunit physically interacted with one another. The expression and secretion of retinoschisin are under circadian regulation with a peak at night and nadir during the day. Inhibition of L-type VGCCs decreased membrane-bound retinoschisin at night. Overexpression of a missense RS1 mutant gene, R141G, into chicken cone photoreceptors caused a decrease of L-type VGCC currents at night. Our findings demonstrate a novel bidirectional relationship between an ion channel and an extracellular protein; L-type VGCCs regulate the circadian rhythm of retinoschisin secretion, whereas secreted retinoschisin feeds back to regulate L-type VGCCs. Therefore, physical interactions between L-VGCCα1 subunits and retinoschisin play an important role in the membrane retention of L-VGCCα1 subunits and photoreceptor-bipolar synaptic transmission

Nifedipine reveals the existence of two discrete components of the progesterone-induced [Ca2+]i transient in human spermatozoa

AbstractThe steroid progesterone, an agonist of acrosome reaction, induces a biphasic [Ca2+]i-signal in human sperm comprising an initial transient [Ca2+]i elevation, and a subsequent ramp or plateau. Nifedipine, an inhibitor of voltage-operated Ca2+ channels, inhibits progesterone-induced acrosome reaction in human sperm, but fluorimetric studies have detected no effect of this compound on the progesterone-induced [Ca2+]i signal. We have used single-cell imaging to study the effects of nifedipine on [Ca2+]i signalling in human sperm. Analysis of mean responses from large numbers of cells showed that treatment with nifedipine reduced the duration but not the amplitude of the progesterone-induced [Ca2+]i transient. In control cells, the latency of the transient peak (maximum fluorescence) fell within the range of 30–105 s. In the presence of nifedipine, very few cells peaked “late” (>60 s after application of progesterone). Analysis of transient responses in control cells revealed characteristic “early” and “late” responses, most cells showing both “early” and “late” transients, whereas “late” transients were rare and smaller in the presence of nifedipine. Sustained responses showed strong association with late transients, and occurrence and amplitude of sustained responses were significantly reduced in nifedipine pretreated cells.These findings are consistent with the occurrence of a discrete, nifedipine-sensitive component of the progesterone-induced [Ca2+]i transient that peaks 1–2 min after exposure to the hormone and is crucial for the induction of the sustained [Ca2+]i signal

Regulation of the hyperpolarization-activated cationic current I(h) in mouse hippocampal pyramidal neurones by vitronectin, a component of extracellular matrix

Because the hyperpolarization-activated cation-selective current I(h) makes important contributions to neural excitability, we examined its long-term regulation by vitronectin, an extracellular matrix component commonly elevated at injury sites and detected immunochemically in activated microglia. Focusing on mouse hippocampal pyramidal neurones in organotypic slice cultures established at postnatal day 0 or 1 and examined after 3–4 days in vitro, we observed differences in the amplitude and activation rate of I(h) between neurones in naive and vitronectin-exposed slices (10 μg ml(−1) added to serum-free medium), and between neurones in slices derived from wild-type and vitronectin-deficient mice. The potassium inward rectifier I(K(ir)), activated at similar voltages to I(h), was not affected by vitronectin. In CA1, differences in I(h) amplitude primarily reflected changes in maximum conductance (G(max)): a 23.3% increase to 3.18 ± 0.64 nS from 2.58 ± 0.96 nS (P < 0.05) in vitronectin-exposed neurones, and a 17.9% decrease to 2.24 ± 0.26 nS from 2.73 ± 0.64 nS (P < 0.05) in neurones from vitronectin-deficient slices. The voltage of one-half maximum activation (V(½)) was not significantly affected by vitronectin exposure (−78.1 ± 2.3 mV versus −80.0 ± 4.9 mV in naive neurones; P > 0.05) or vitronectin deficiency (−83.8 ± 3.1 mV versus −82.0 ± 2.9 mV in wild-type neurones; P > 0.05). In CA3 neurones, changes in I(h) reflected differences in both G(max) and V(½): in vitronectin-exposed neurones there was a 35.4% increase in G(max) to 1.30 ± 0.49 nS from 0.96 ± 0.26 nS (P < 0.01), and a +3.0 mV shift in V(½) to −89.8 mV from −92.8 mV (P < 0.05). The time course of I(h) activation could be fitted by the sum of two exponential functions, fast and slow. In both CA1 and CA3 neurones the fast component amplitude was preferentially sensitive to vitronectin, with its relatively larger contribution to total current in vitronectin-exposed cells contributing to the acceleration of I(h) activation. Further, HCN1 immunoreactivity appeared elevated in vitronectin-exposed slices, while HCN2 levels appeared unaltered. We suggest that vitronectin-stimulated increases in I(h) may potentially affect excitability under pathological conditions