Electrogenic Na(+)/Ca(2+) exchange. A novel amplification step in squid olfactory transduction

Abstract

Olfactory receptor neurons (ORNs) from the squid, Lolliguncula brevis, respond to the odors l-glutamate or dopamine with increases in internal Ca(2+) concentrations ([Ca(2+)](i)). To directly asses the effects of increasing [Ca(2+)](i) in perforated-patched squid ORNs, we applied 10 mM caffeine to release Ca(2+) from internal stores. We observed an inward current response to caffeine. Monovalent cation replacement of Na(+) from the external bath solution completely and selectively inhibited the caffeine-induced response, and ruled out the possibility of a Ca(2+)-dependent nonselective cation current. The strict dependence on internal Ca(2+) and external Na(+) indicated that the inward current was due to an electrogenic Na(+)/Ca(2+) exchanger. Block of the caffeine-induced current by an inhibitor of Na(+)/Ca(2+) exchange (50-100 microM 2',4'-dichlorobenzamil) and reversibility of the exchanger current, further confirmed its presence. We tested whether Na(+)/Ca(2+) exchange contributed to odor responses by applying the aquatic odor l-glutamate in the presence and absence of 2', 4'-dichlorobenzamil. We found that electrogenic Na(+)/Ca(2+) exchange was responsible for approximately 26% of the total current associated with glutamate-induced odor responses. Although Na(+)/Ca(2+) exchangers are known to be present in ORNs from numerous species, this is the first work to demonstrate amplifying contributions of the exchanger current to odor transduction.

Figure 1

10 mM caffeine activates a sodium-dependent current in nystatin-perforated patched squid ORNs. (A) Current trace from a nystatin-perforated patched ORN during a 1-s application of 10 mM caffeine. Solutions ASW external (ext)/TMA Cl internal (int). (B) In the same cell, replacing external Na⁺ with Tris⁺ completely eliminated the caffeine-induced current. Solutions: Tris ASW ext/TMA Cl int. (C) A caffeine-activated current response in ASW from a different cell. Same solutions as in A. (D) Current trace from the cell in C in Li⁺ ASW. (E) Average current density of caffeine-induced current responses from squid ORNs in ASW, Tris⁺ ASW, ASW, and Li⁺ ASW, respectively. The holding potential has been corrected for liquid junction potential and is indicated in each panel. Error bars indicate SD. *P < 0.05.

Figure 3

Caffeine-induced currents are delayed relative to odor-induced currents. (A) This current trace was obtained from a nystatin-perforated patched squid ORN during a 1-s application of 5 mM glutamate. (B) In the same cell, the current response to a 1-s application of 10 mM caffeine is markedly delayed. Solutions: ASW ext/TMA gluconate int.

Figure 2

DCB reversibly eliminates Na⁺/Ca²⁺ exchange in squid ORNs. (A) 10 mM caffeine elicits a large Na⁺/Ca²⁺ exchange current. (B) After 12 s in the presence of 100 μM DCB, I_NCX is partially blocked and, after 42 s, I_NCX is completely eliminated. (C) After removing DCB and allowing the cell to recover for 2 min, I_NCX is restored. (D) In all 18 cells tested, DCB eliminated I_NCX. Solutions: ASW ext./Na gluconate int. *P < 0.001.

Figure 4

Na⁺/Ca²⁺ exchange currents are reversible in squid ORNs. (A) Current traces from a squid ORN under whole-cell voltage-clamp conditions. The solid line above the current trace indicates the switch from ASW to Tris⁺ ASW. +, 20-mV hyperpolarization. External and internal sodium concentrations are noted above and below the solid line in millimoles per liter⁻¹. Solutions: ASW ext (Tris⁺ ASW)/Na⁺ gluconate int with 4 mM Mg²⁺ and 2 mM ATP. (B) A different ORN exposed to the same protocol as in A, but with Li⁺ gluconate internal solution to eliminate reverse Na⁺/Ca²⁺ exchange. The solid line indicates the switch from ASW to Tris⁺ ASW and back again. Numbers indicate external and internal sodium concentrations in millimoles per liter⁻¹. +, 20-mV hyperpolarizations. Solutions: ASW ext (Tris⁺ ASW)/Li⁺ gluconate int; R_s = 19 MΩ, equilibration time = 5 min). (C) Mean percent decrease in the steady state inward current measured during the switch to Tris ASW under the conditions in A (Na⁺ int) and B (Li⁺ int). *The mean current reduction in Li⁺ gluconate is significantly smaller than Na⁺ gluconate int P < 0.05. Error bars indicate SD; n = numbers of cells.

Figure 5

I_NCX amplifies odorant responses in squid ORNs. (A) Three successive responses to 5 mM glutamate, 5 mM glutamate in 50 μM DCB, and 5 mM glutamate, respectively, are shown for a nystatin-perforated patched squid ORN. This cell was exposed to DCB for 36 s before application of glutamate + DCB. When DCB is removed, the size of the glutamate-induced current returns to control levels. (B) The magnitude of the peak current was normalized to the peak of the control responses and plotted as a bar graph. DCB was either coapplied with glutamate (n = 8) or pre-incubated for 58 ± 21 s (n = 10). The combined DCB data show that the response to 5 mM glutamate is reduced by 26 ± 10% (n = 18; vertical stripes). In the absence of external Na⁺ (Li⁺ + Glu, diagonal stripes), 5 mM glutamate responses were reduced by 31 ± 7%. Error bars indicate SD. *P < 0.05. Solutions: ASW ext/60 TEA Na gluconate int.

Figure 6

DCB has no effect on glutamate-induced currents in Li⁺ ASW. (A) Current trace from a nystatin-perforated patched squid ORN treated with 5 mmol liter⁻¹ glutamate in Li⁺ ASW. (B) Current trace from the same cell in the presence of 50 μM DCB. Note that the magnitude of the current is unchanged. (C) Bar graph comparing the average, normalized current responses to glutamate from squid ORNs in Li⁺ ASW (crosshatch) and in Li⁺ ASW + 50 μM DCB (diagonal stripes). In the presence of DCB, responses to glutamate were 98 ± 14% of the control glutamate responses in Li⁺ ASW, and were not significantly different from each other (P > 0.05). Error bar indicates SD. Solutions: Li⁺ ASW ext/60 TEA Na⁺ gluconate int.