Giorgio Rispoli

Calcium Currents in Hair Cells Isolated from Semicircular Canals of the Frog

L-type and R-type Ca(2+) currents were detected in frog semicircular canal hair cells. The former was noninactivating and nifedipine-sensitive (5 microM); the latter, partially inactivated, was resistant to omega-conotoxin GVIA (5 microM), omega-conotoxin MVIIC (5 microM), and omega-agatoxin IVA (0.4 microM), but was sensitive to mibefradil (10 microM). Both currents were sensitive to Ni(2+) and Cd(2+) (>10 microM). In some cells the L-type current amplitude increased almost twofold upon repetitive stimulation, whereas the R-type current remained unaffected. Eventually, run-down occurred for both currents, but was prevented by the protease inhibitor calpastatin. The R-type current peak component ran down first, without changing its plateau, suggesting that two channel types generate the R-type current. This peak component appeared at -40 mV, reached a maximal value at -30 mV, and became undetectable for voltages > or =0 mV, suggestive of a novel transient current: its inactivation was indeed reversibly removed when Ba(2+) was the charge carrier. The L-type current and the R-type current plateau were appreciable at -60 mV and peaked at -20 mV: the former current did not reverse for voltages up to +60 mV, the latter reversed between +30 and +60 mV due to an outward Cs(+) current flowing through the same Ca(2+) channel. The physiological role of these currents on hair cell function is discussed.

Enhanced dihydropyridine receptor calcium channel activity restores muscle strength in JP45/CASQ1 double knockout mice

Muscle strength declines with age in part due to a decline of Ca(2+) release from sarcoplasmic reticulum calcium stores. Skeletal muscle dihydropyridine receptors (Ca(v)1.1) initiate muscle contraction by activating ryanodine receptors in the sarcoplasmic reticulum. Ca(v)1.1 channel activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Ca(v)1.1 and the sarcoplasmic reticulum Ca(2+) storage protein calsequestrin (CASQ1). Here we show that JP45 and CASQ1 strengthen skeletal muscle contraction by modulating Ca(v)1.1 channel activity. Using muscle fibres from JP45 and CASQ1 double knockout mice, we demonstrate that Ca(2+) transients evoked by tetanic stimulation are the result of massive Ca(2+) influx due to enhanced Ca(v)1.1 channel activity, which restores muscle strength in JP45/CASQ1 double knockout mice. We envision that JP45 and CASQ1 may be candidate targets for the development of new therapeutic strategies against decay of skeletal muscle strength caused by a decrease in sarcoplasmic reticulum Ca(2+) content.

Calcium‐activated potassium current clamps the dark potential of vertebrate rods

Vertebrate photoreceptors respond to light with a graded hyperpolarization from a membrane potential in the dark of ≈ −35 mV. The present work investigates the physiological role of the Ca2+‐activated K+ current in the photovoltage generation in mechanically isolated rods from salamander retina. Membrane current or voltage in isolated rods was recorded from light‐ and dark‐adapted rods under voltage‐ or current‐clamp conditions, respectively. In light‐adapted rods of the salamander, selective blockade of Ca2+‐activated K+ channels by means of charybdotoxin depolarized the plasma membrane of current‐clamped rods by ≈ 30 mV, from a resting potential of ≈ −35 mV. A similar depolarization was observed if external Ca2+ (1 mm) was substituted with Ba2+ or Sr2+. Under control conditions, the injection of currents of increasing amplitude (up to −100 pA, to mimic the current entering the rod outer segment) could not depolarize the membrane potential beyond a saturating value of ≈ −20 mV. However, in the presence of charybdotoxin, rods depolarized up to +20 mV. In experiments with dark‐adapted current‐clamped rods, charybdotoxin perfusion lead to transient depolarizations up to 0 mV and steady‐state depolarizations of ≈ 5 mV above the dark resting potential. Finally, the recovery phase of the voltage response to a flash of light in the presence of charybdotoxin showed a transient overshoot of the membrane potential. It was concluded that Ca2+‐activated K+ current is necessary for clamping the rod photovoltage to values close to the dark potential, thus allowing faithful single photon detection and correct synaptic transmission.

Transport of K+ by Na(+)-Ca2+, K+ exchanger in isolated rods of lizard retina.

Transport of K+ by the photoreceptor Na(+)-Ca2+, K+ exchanger was investigated in isolated rod outer segments (OS) by recording membrane current under whole-cell voltage-clamp conditions. Known amounts of K+ were imported in the OS through the Ca(2+)-activated K+ channels while perfusing with high extracellular concentration of K+, [K+]o. These channels were detected in the recordings from the OS, which probably retained a small portion of the rest of the cell. The activation of forward exchange (Na+ imported per Ca2+ and K+ extruded) by intracellular K+, Ki+, was described by first-order kinetics with a Michaelis constant, Kapp(Ki+), of about 2 mM and a maximal current, Imax, of about -60 pA. [Na+]i larger than 100 mM had little effect on Kapp(Ki+) and Imax, indicating that Nai+ did not compete with Ki+ for exchange sites under physiological conditions, and that Na+ release at the exchanger intracellular side was not a rate-limiting step for the exchange process. Exchanger stoichiometry resulted in one K+ ion extruded per one positive charge imported. Exchange current was detected only if Ca2+ and K+ were present on the same membrane side, and Na+ was simultaneously present on the opposite side. Nonelectrogenic modes of ion exchange were tested taking advantage of the hindered diffusion found for Cai2+ and Ki+. Experiments were carried out so that the occurrence of a putative nonelectrogenic ion exchange, supposedly induced by the preapplication of certain extracellular ion(s), would have resulted in the transient presence of both Cai2+ and Ki+. The lack of electrogenic forward exchange in a subsequent switch to high Nao+, excluded the presence of previous nonelectrogenic transport.

Regional distribution of calcium currents in frog semicircular canal hair cells

In the present work we studied the regional expression of voltage-dependent Ca channels in hair cells from the frog semicircular canals, employing whole-cell patch-clamp on isolated and in situ hair cells. Although Ca channels are thought to play a major role in afferent transmission, up to now no data were available regarding their distribution in vestibular organs. The problem appears of interest, especially in the light of recent results showing the presence of multiple Ca current components in semicircular canal hair cells. Our data suggest the presence, in all regions of the crista ampullaris, of two classes of cells, one displaying an inactivating Ca current (R1) and one lacking it. In the former cells, Ca current amplitude decreased from the central to the peripheral zone (the maximal currents being observed in the intermediate zone). Only L-type and R2 current components displayed regional differences in expression, whereas the size and properties of R1, although variable among cells, were not regionalized. However, in cells lacking R1, Ca current amplitudes were similar regardless of cell shape and location. The possible contributions of this Ca current distribution to afferent discharge properties are discussed.

IP3 receptor in the hair cells of frog semicircular canal and its possible functional role.

The presence and functional role of inositol trisphosphate receptors (IP3R) was investigated by electrophysiology and immunohistochemistry in hair cells from the frog semicircular canal. Intracellular recordings were performed from single fibres of the posterior canal in the isolated, intact frog labyrinth, at rest and during rotation, in the presence of IP3 receptor inhibitors and drugs known to produce Ca2+ release from the internal stores or to increase IP3 production. Hair cell immunolabelling for IP3 receptor was performed by standard procedures. The drug 2‐aminoethoxydiphenyl borate (2APB), an IP3 receptor inhibitor, produced a marked decrease of mEPSP and spike frequency at low concentration (0.1 mm), without affecting mEPSP size or time course. At high concentration (1 mm), 2APB is reported to block the sarcoplasmic‐endoplasmic reticulum Ca2+‐ATPase (SERCA pump) and increase [Ca2+]i; at the labyrinthine cytoneural junction, it greatly enhanced the resting and mechanically evoked sensory discharge frequency. The selective agonist of group I metabotropic glutamate receptors (RS)‐3,5‐dihydroxyphenylglycine (DHPG, 0.6 mm), produced a transient increase in resting mEPSP and spike frequency at the cytoneural junction, with no effects on mEPSP shape or amplitude. Pretreatment with cyclopiazonic acid (CPA, 0.1 mm), a SERCA pump inhibitor, prevented the facilitatory effect of both 2APB and DHPG, suggesting a link between Ca2+ release from intracellular stores and quantal emission. Consistently, diffuse immunoreactivity for IP3 receptors was observed in posterior canal hair cells. Our results indicate the presence and a possibly relevant functional role of IP3‐sensitive stores in controlling [Ca2+]i and modulating the vestibular discharge.

A step-by-step model of phototransduction cascade shows that Ca2+ regulation of guanylate cyclase accounts only for short-term changes of photoresponse

A mathematical model of the vertebrate phototransduction mechanism was designed in a modular fashion, in that increasingly complex behaviors can be turned on and off to evaluate the relative involvement of all elements of the phototransduction cascade. The problem was approached by starting with a minimum model in which the intracellular cGMP concentration ([cGMP]i) was determined by guanylate cyclase (GC), whose activity was assumed not to be regulated by any factor (such as Ca2+) and by phosphodiesterase (PDE), whose activity was assumed to be proportional to the light intensity. All dependences were subsequently introduced, i.e. the equations describing PDE activation in detail, the Ca2+ regulation of GC and the action of intracellular Ca2+ buffers. The simulations and fits show that a high-gain, smooth time- and light-dependent PDE activation, a Ca2+-dependent GC, and a Ca2+-dependent buffer mechanism are required to account for the flash response kinetics in the dark and on dim backgrounds of light, and the effect of exogenous Ca2+ buffers to produce responses characterized by slow and damped oscillations and to enhance the low-frequency noise. However, it was not possible to find any set of parameters able to simultaneously interpolate the waveform of the flash responses (in the dark and on a background of light) and the responses to steps of light. It is therefore concluded that at least one more shut-off mechanism (possibly not Ca-dependent) is necessary to fully account for the phenomenology of the light response in rod photoreceptors.

Electrophysiological characterization of ionic transport by the retinal exchanger expressed in human embryonic kidney cells

The retinal Na+:Ca2+, K+exchanger cDNA was transiently expressed in human embryonic kidney (HEK 293) cells by transfection with plasmid DNA. The correct targeting of the expressed protein to the plasma membrane was confirmed by immunocytochemistry. The reverse exchange offrent (Ca2+ imported per Na+ extruded) was measured in whole-cell voltage-clamp experiments after intracellular perfusion with Na+ (Na+i, 128 mM) and extracellular perfusion with Ca2+ (Ca2o+, 1 mM) and Ko+ (20 mM). As expected, the exchange current was suppressed by removing Ca2o+. Surprisingly, however, it was also abolished by increasing Na+o to almost abolish the Na+ gradient, and it was almost unaffected by the removal of Ko+. Apparently, then, at variance with the exchanger in the rod outer segment, the retinal exchanger expressed in 293 cells acts essentially as a Na+:Ca2+ exchanger and does not require K+ for its electrogenic activity.

Pore Forming Properties of Cecropin-Melittin Hybrid Peptide in a Natural Membrane

The pore forming properties of synthetic cecropin-melittin hybrid peptide (Acetyl-KWKLFKKIGAVLKVL-CONH2; CM15) were investigated by using photoreceptor rod outer segments (OS) isolated from frog retinae obtained by using the whole-cell configuration of the patch-clamp technique. CM15 was applied (and removed) to (from) the OS in ~50 ms with a computer-controlled microperfusion system. Once the main OS endogenous conductance was blocked with light, the OS membrane resistance was ≥1 GΩ, allowing high resolution, low-noise recordings. Different to alamethicines, CM15 produced voltage-independent membrane permeabilisation, repetitive peptide application caused a progressive permeabilisation increase, and no single-channel events were detected at low peptide concentrations. Collectively, these results indicate a toroidal mechanism of pore formation by CM15.

Divalent cations modulate membrane binding and pore formation of a potent antibiotic peptide analog of alamethicin

The Ca(2+) modulation of pore formation (and disaggregation) kinetics of a synthetic analog of alamethicin F50/5 ([l-Glu(OMe)(7,18,19)]), a potent antibiotic peptide, was investigated in situ and in vitro. The in situ experiments consisted in whole-cell recording from isolated retinal rod outer segments (OS), because once blocking the only OS endogenous conductance with saturating light, the current flows entirely through the (exogenous) channels formed by the peptide. The kinetics of current change induced by peptide application and removal (in ∼50ms) on the OS extracellular side was measured in the presence of divalent cations at different concentrations. The in vitro experiments consisted on the divalent cations modulation of [l-Glu(OMe)(7,18,19)] binding to a mimetic OS membrane immobilized on a sensor chip surface, employing surface plasmon resonance spectroscopy (SPR). The presence of even low mM Ca(2+) or Mg(2+) sufficed to increase the [l-Glu(OMe)(7,18,19)] apparent affinity for the mimetic OS membrane up to ∼4-fold, which accelerated the activation of the peptide-induced current in OS by ∼10-fold with respect to low Ca(2+). In situ and in vitro experiments indicate that high concentrations of divalent cations increased also membrane rigidity, contrasting their effect on increasing the pore formation rate.