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Calcium currents from jellyfish striated muscle cells: preservation of phenotype, characterisation of currents and channel localisation

Y.-C. James Lin and Andrew N. Spencer^*

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 and Bamfield Marine Station, Bamfield, British Columbia, Canada V1R 1B0

View larger version (56K): [in a new window]   Fig. 1. Morphology of striated muscle cells dissociated from Polyorchis penicillatus at different temperatures and their associated membrane currents. (A) Phase-contrast micrograph of muscle cells dissociated at 20–22 °C showing that cells lose muscle feet and round up. Arrow indicates one tear-shaped cell with a remnant of myofibres. (B) Phase-contrast micrograph of muscle cells dissociated at 30 °C showing a muscle cell with two pairs of feet and a patch-recording pipette attached. (C) Histogram to show that muscle cells dissociated with 1 mg ml-1 Pronase at 30 °C retain their in situ morphology with 84.51±6.80 % of cells having two or more pairs of muscle feet (myofibres). Values are means ± S.E.M. (D) Voltage-clamp recording of total membrane currents from a cell dissociated at 20–22 °C showing that both inward and outward current amplitudes are low. The stimulus protocol was 25 ms test pulses, incrementing in 10 mV steps from -70 to +90 mV from a holding potential of -80 mV. (E) Voltage-clamp recording of total membrane currents from a cell dissociated at 30 °C showing increased amplitude of the currents. Stimulus protocol as for D.

View larger version (21K): [in a new window]   Fig. 2. Low-voltage-activated calcium current recorded from a muscle cell dissociated at 30 °C. (A) Current traces recorded in whole-cell, voltage-clamp mode using a bath solution where calcium was replaced by 50 mmol l-1 SrCl2 to prevent run-down of this current. All potassium currents were blocked by CsOH, CsCl2 and TEA-Cl in the pipette solution (see Materials and methods). The stimulus protocol was a series of 25 ms test potentials with increments of 10 mV from -70 to +90 mV using a holding potential of -80 mV. (B) Mean I/V curve from 10 cells using the above protocols. Values are means ± S.E.M.

View larger version (14K): [in a new window]   Fig. 3. Voltage dependence of time-to-peak (A) and inactivation time constant (; B) of the low-voltage-activated calcium current. Sr2+ was used as the charge carrier. The stimulus protocol for both graphs was a series of 25 ms test pulses with increments of 10 mV from -70 to +90 mV from a holding potential of -80 mV. The time constant () for the inactivation decay was obtained by fitting the calcium current decay with a single exponent at each testing potential using the Clampfit program (Axon Instruments). The means from each testing potential were fitted with Sigma Plot 4.0 program (SPSS Inc.). Values are means ± S.E.M., N=10 cells.

View larger version (14K): [in a new window]   Fig. 4. Steady-state inactivation and recovery from inactivation of the low-voltage-activated calcium current. Sr2+ was used as the charge carrier. (A) The steady-state inactivation curve was obtained from data generated using 20 ms test pulses to -30 mV immediately following 2 s conditioning prepulses in 5 mV increments from -110 to -30 mV. The holding potential prior to the conditioning pulse was -80 mV. Steady-state inactivation data were normalised to averaged maximal current (I/Imax) and the means fitted with a Boltzman equation (I/Imax=1/{1+exp[(Vpp-V50)/k]}, where Vpp is the prepulse voltage, V50 is the prepulse voltage causing half-inactivation; k is the slope factor of the inactivation curve in mV per e-fold change). V50=-80±3.5 mV, k=7.7±1.9 mV, N=7. (B) The curve for recovery from inactivation was generated from data obtained using 20 ms inactivating prepulses to -30 mV from a holding potential of -80 mV, followed by a recovery period (Rt) of variable duration from 6.4 to 800 ms at -80 mV, and a 25 ms test pulse to -30 mV. Peak currents obtained in response to test pulses were normalised to prepulse values (I/Imax) and fitted with exponential curves (I/Imax=1-exp(-Rt/), where Rt is the recovery period and is the time constant). =51.7±0.8 ms. Values are means ± S.E.M., N=10.

View larger version (21K): [in a new window]   Fig. 5. Effect of various dihydropyridines and verapamil on muscle contraction. Velar strips were subjected to field stimulation at 0.2 Hz for 30 ms. Mean peak tension was registered on a digital pen recorder through a capacitative force transducer. Peak tension was normalised to that in the ASW control. The perifusion rate was set at 1.5 ml min-1 and the temperature of the perifusate was 10–12 °C. Values are means ± S.E.M.

View larger version (24K): [in a new window]   Fig. 6. Effect of dihydropyridines, verapamil and NiCl2 on the low-voltage activated calcium current of muscle cells. Sr2+ was used as the charge carrier. Unmarked traces are those recorded before the drug was applied while traces marked with an asterisk indicate those recorded with the drug applied, and traces marked by an arrow indicate those recorded after washing with bath solution. The stimulus protocol used was a single 20 ms voltage step to -30 mV from a holding potential of -80 mV. The early large phasic negative-going current excursion is the uncompensated capacitative current while the transients at the end of the traces are the ‘off’ capacitative transients.

View larger version (30K): [in a new window]   Fig. 7. Localisation of calcium channels using dihydropyridine-BODIPY (fDHP) and RH414 in striated muscles dissociated at room temperature (20°C; A) and at 30 °C (B). Dissociated cells were incubated with both fDHP (final concentration 10 µmol l-1) and the styryl dye RH414 (final concentration 5 µmol l-1), for 15 min at room temperature before examination. Images were obtained using laser-scanning, confocal microscopy. The wavelengths of the excitation beam were set to 488 nm and 568 nm. The emitted fluorescence from labelled cells was collected at 530 nm for green fluorescence (fDHP) and 590 nm for red fluorescence (RH414). At 20 °C cells were spherical and fDHP labelled mostly small vesicles within the cytoplasm (A) while at 30 °C there was strong fDHP labelling of the sarcolemma of the muscle feet (B). Scale bars, 2 µm. (C) Ratio of labelling intensity of green (fDHP) versus red (RH414) fluorescence in the plasma membrane of myofibres (feet) and the cell somata. Data on the intensity of fluorescence (both green and red) were collected from those areas of the membrane showing sharp and definitive RH414 labelling (red), indicating that a perpendicular optical section through the cell membrane was being analyzed. The intensity of green fluorescence (fDHP) was divided by the intensity of red fluorescence (RH414) pixel-by-pixel using NIH image software. Values are means ± S.E.M., N=20 cells. Asterisk, significant difference (P<0.05).