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First published online June 29, 2006
Journal of Experimental Biology 209, 2749-2764 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02312

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Dopamine modulation of Ca²⁺ dependent Cl^- current regulates ciliary beat frequency controlling locomotion in Tritonia diomedea

Owen M. Woodward^1,2,* and A. O. Dennis Willows^1,2

¹ Department of Biology, University of Washington, Seattle, WA 98195, USA
² Friday Harbor Laboratories, University of Washington, 620 University Road, Friday Harbor, WA 98250, USA

View larger version (17K): [in a new window]   Fig. 1. Whole cell currents of ciliated pedal epithelial cells for Tritonia diomedea. (A) Ionic currents resulting from voltage clamp depolarizing steps in a representative cell show large, slowly activating and voltage dependent currents. The slow I-V (B) reveals a strong voltage dependence. The initial current size is not dependent on the inclusion of 2 mmol l-1 ATP and 100 µmol l-1 cAMP in the recording pipette internal solution (open triangles, with ATP and cAMP, N=50; filled circles, without, N=76).

View larger version (57K): [in a new window]   Fig. 2. In situ patch method allows for successful recordings from ciliated epithelial cells. (Ai) Representative micrograph taken while recording using the in situ patch technique. Identifying whether the cell being recorded from is ciliated is not possible. Scale bar, 25 µm. (Aii) Representative voltage clamp records of currents elicited from eight +20 mV step increases beginning from -57 mV. (Bi) Representative micrograph taken while recording from a disassociated epithelia cell that can be clearly identified as ciliated. Scale bar, 25 µm. (Bii) Voltage clamp records from cell pictured in Bi of currents elicited from eight +20 mV step increases beginning from -57 mV. (C) Slow current/voltage relationship reveals no difference in kinetics or amplitude of currents recorded using the `blind' patch technique (circles, N=76) or from disassociated clearly identified ciliated cells (triangles, N=12).

View larger version (22K): [in a new window]   Fig. 3. Large portion of whole cell current is carried by Cl- ions. (A) Representative voltage clamp records of currents (elicited from seven +20 mV step increases, beginning from -60 mV) from a cell in low (132 mmol l-1) Cl- bath solution (shadow, a representative trace from a different cell in ASW at +63 mV). Open arrowhead, position of fast I-V; filled arrowhead, position of slow I-V. (B) Slow I-V reveals the cells in the low Cl- bath (triangles, N=18) have substantially reduced currents as compared to those in normal ASW (circles, N=76). (B,inset) Comparison of maximum current sizes between ASW (73 mV; N=76) and Low Cl- (70 mV; N=18) reveal a significantly smaller current amplitude for those cells in a Low Cl- bath (P=2.4x10-11). (C) Fast I-V shows an even larger difference in current amplitude (circles, ASW; N=76; triangles, Low Cl-; N=18). (C,inset) Comparison of current amplitudes after 40 ms of depolarization show a larger discrepancy in current amplitude between ASW at 73 mV and Low Cl- at 70 mV (P=3.4x10-11), suggesting that the Cl- current is the faster activating current. (D) Whole cell current amplitude is significantly reduced after the application of Cl- channel blockers DIDS and 9-AC (500 µmol l-1 DIDS P=0.01, N=4; 50 µmol l-1 9-AC P=0.01, N=4).

View larger version (17K): [in a new window]   Fig. 4. The whole cell current reversal potential shifts in the low Cl- bath in a predictable fashion. Representative voltage clamp records of tail currents in ASW (A), and in Low Cl- (B). (C) I-V graph of the instantaneous tail currents from A and B, demonstrating a shift in the reversal potential. The mean observed shift (difference between the mean ASW Erev, N=13, and Low Cl-, Erev N=9) is +34.7 mV. The predicted shift is +33.2 mV.

View larger version (22K): [in a new window]   Fig. 5. Exponential analysis of whole cell currents reveals two conductances. (A) Exponential fits on representative currents elicited from a 60 mV (Low Cl-) or 63 mV (ASW). The ASW trace was successfully fit with a double exponential, and the Low Cl- trace could be fit successfully with a single exponential (B). Examination of the amplitudes and time constants for each current (fast and slow) (B) reveals that in the Low Cl- bath the fast current disappears.

View larger version (30K): [in a new window]   Fig. 6. The fast Cl- currents show Ca2+ dependence. (A) Representative voltage clamp records of currents (elicited from eight +20 mV step increases, beginning from -57 mV) from a cell in a zero Ca2+ bath solution (shadow, a representative trace from a different cell in ASW at +63 mV). Open arrowhead, position of fast I-V; filled arrowhead, position of slow I-V. (Bi) Slow I-V reveals the cells in the 0 Ca2+ bath (open squares, N=5) have reduced currents as compared to those in normal ASW (filled circles, N=76), but are larger than the currents in a Low Cl- bath (open triangles, N=18). Smaller still are currents from cells in a Low Cl-, 0 Ca2+ bath (open diamonds, N=10). (Bii) Comparison of maximum current amplitude between ASW (at 73 mV, N=76), 0 Ca2+ (73 mV, N=5), Low Cl- (70 mV, N=18), and Low Cl-, 0 Ca2+ (70 mV, N=10) reveal each treatment results in significantly smaller current amplitude when compared to the ASW control (P=8.7x10-15). (Ci) Fast I-V reveals an even larger difference in current amplitude between ASW (filled circles, N=76), and the 0 Ca2+ bath (open squares, N=5), which remains slightly larger than the currents in a Low Cl- bath (open triangles, N=18). (Cii) Comparison of maximum current shows an even larger discrepancy in amplitude between ASW (at 73 mV, N=76) and 0 Ca2+ bath (73 mV, N=5) and Low Cl- (70 mV, N=18) (P=3.4x10-11). (D) Currents from one cell depolarized to 63 mV before and after the addition of 2 mmol l-1 Cd2+ to the bath. (E) Currents from one cell depolarized to 63 mV before and after the addition of the Ca2+ channel blocker nifedipine (Nif; 50 µmol l-1). (F) Means for the total current reduction after exposure to either nifedipine or Cd2+. Both the Cd2+ reduction (N=7, P=0.01) and the nifedipine reduction (N=5, P=1.9x10-5) are significant.

View larger version (28K): [in a new window]   Fig. 7. Blockage of Cl- currents reveals a second voltage dependent current. (Ai) In a bath containing 0 Cl- and 0 Ca2+, slow outward voltage dependent currents persist (shadow, a representative trace from a different cell in ASW at +63 mV). The activation curve of the currents can be fit successfully with a Boltzman function (Aii) (correlation=0.949, N=7). The high K value equates roughly to 1.2 charges, assuming the simple open-close model for channel activation. Fast I-V (Aiii) shows 0 Cl-, 0 Ca2+ bath (triangles) removes completely the fast Cl- current component (compared to ASW bath, circles), leaving only the slow activating putative H+ current. The currents are very sensitive to the application of zinc. (B) The currents from a representative cell before (Bi) and after (Bii) the application of 10 µmol l-1 ZnCl2. A slow I-V plot (Biii) shows that amplitudes at all voltages are dramatically reduced in the presence of ZnCl2 (N=7). Finally, decreasing pH causes a reduction in current amplitude and a shift in the current reversal potential. (Ci) Currents from a representative cell show a decrease in amplitude in the lower 7.0 pH and (Cii) a plot of the shift in reversal potential Erev from the same cell shows a shift of +52 mV. The mean shift measured was +51.867±6.7 mV (N=5), very close to the predicted +56 mV shift.

View larger version (23K): [in a new window]   Fig. 8. Dopamine increases CBF. (A) Raw voltage recordings from transducer measuring CBF. Each oscillation represents a cilium passing in front of the transducer. Dopamine increases the rate of oscillation and therefore CBF. (B) Dose-response for concentrations of dopamine applied to CPE cell explants show a maximal excitatory effect on CBF around 100 µmol l-1. High concentrations (10 mmol l-1, N=6) and low concentrations (1 µmol l-1, N=5) did not change CBF. The middle concentrations all show significant increases in CBF compared to seawater controls (10 µmol l-1 N=5, P=0.0001; 100 µmol l-1 N=5, P=0.0003; 1 mmol l-1 N=6, P=0.0001). (C) Dopamine causes a significant increase in the CBF (N=5, P=0.0002) of isolated CPE cells.

View larger version (23K): [in a new window]   Fig. 9. Dopamine and TPep-NLS decrease total whole cell current amplitude. (A) Whole cell current recordings from CPE cells in a seawater bath. (B) Same cell after exposure to 100 µmol l-1 dopamine. (C) Normalized I-V 900 ms into voltage step reveals that dopamine (DA) reduces current amplitude at all voltages that the currents are activated (before dopamine, circles; after dopamine, squares; N=7). (C,inset) Dopamine caused a significant decrease in maximum current amplitude at 73 mV (N=7, P<0.001). (D) Whole cell current recordings from CPE cells in a seawater bath. (E) Same cell after exposure to 10 µmol l-1 TPep-NLS. (F) Normalized I-V 900 ms into voltage step reveals that TPep-NLS reduces current amplitude at all voltages the currents are activated (before TPep-NLS, squares; after TPep-NLS, circles; N=7). (F,inset) TPep-NLS caused a significant decrease in maximum current amplitude at 73 mV (N=10, P<0.001).

View larger version (18K): [in a new window]   Fig. 10. Dopamine and TPep-NLS selectively reduce the Cl- current of CPE cells. (A) Representative current traces and their double exponential fits from one cell before and after exposure to 100 µmol l-1 dopamine during a 63 mV depolarization. (B) Amplitudes for each of the two exponentials, corresponding to the previously established current types found in CPE cells, before and after exposure to dopamine (N=7). Dopamine selectively lowers the amplitude of the fast activating Cl- current by 55%. (C) Representative current traces and their double exponential fits from one cell before and after exposure to 10 µmol l-1 TPep-NLS during a 63 mV depolarization. (D) Amplitudes for each of the two exponentials, corresponding to the previously established current types found in CPE cells, before and after exposure to TPep-NLS (N=8). TPep-NLS selectively lowers the amplitude of the fast activating Cl- current by 49%.

View larger version (18K): [in a new window]   Fig. 11. Removal of extracellular Cl- or blockage of Cl- currents, cause CBF excitation. 500 µmol l-1 DIDS (N=5), 1 mmol l-1 DIDS (N=5), and zero extracellular Cl- (N=5) each significantly increases CBF in CPE cell explants.

View larger version (25K): [in a new window]   Fig. 12. Model of transmitter and neuropeptide action on CPE cells. Binding of dopamine or TPep-NLS to a receptor leads to a reduction in ICl(Ca) and I(Cl-)leak, leading to an increase in CBF. We hypothesize that I(Cl-)leak is carried by the same CaCC channels as the ICl(Ca), thus a single action by dopamine or TPep-NLS will block both. The blockage of the Cl- currents may lead to a depolarization of the cell, the signal necessary for Ca2+ influx. Pluses indicate molecules or actions that increase current size or beating rate, minuses represent inhibitory molecules or actions.