The effect of external sodium concentration on sodium-calcium exchange in frog olfactory receptor cells

Abstract

During the response of vertebrate olfactory receptor cells to stimulation, Ca(2+) enters the cilia via cyclic nucleotide-gated channels and is extruded by Na(+)-Ca(2+) exchange. The rise in Ca(2+) concentration opens a Ca(2+)-activated Cl(-) conductance which carries most of the inward receptor current. The dependence of Ca(2+) extrusion upon external Na(+) concentration was studied by using the falling phase of the Ca(2+)-activated Cl(-) current following a brief exposure to the phosphodiesterase inhibitor IBMX to monitor indirectly the decay in intraciliary Ca(2+) concentration. External Na(+) concentration was reduced by partial substitution with guanidinium, an ion which permeates the cyclic nucleotide-gated channel but does not support Na(+)-Ca(2+) exchange. The time constant describing the decay in current following IBMX stimulation was surprisingly little affected by substitution of external Na(+), being substantially retarded only when its concentration was reduced to a third or less of its normal value in Ringer solution. When the cilia were returned to Ringer solution after a period in reduced-Na(+) solution, the time constant for the final decay of current was similar to that seen when returning immediately to IBMX-free Ringer solution. This observation suggests that Ca(2+) extrusion via Na(+)-Ca(2+) exchange dominates the falling phase of the response to IBMX, which can therefore be used to assess exchanger activity. Rate constants derived from the time constants for current decay at different external Na(+) concentrations could be fitted by the Hill equation with a K(d) of 54 +/- 4 mm and Hill coefficient of 3.7 +/- 0.4. The cooperativity of the dependence upon external Na(+) concentration indicates that at least three Na(+) ions enter for each exchanger cycle, while the high affinity for external Na(+) contrasts with the photoreceptor and cardiac exchangers. The functional importance of this observation is that the relative insensitivity of the Na(+)-Ca(2+) exchanger to external Na(+) concentration allows normal response termination even following partial dilution or concentration of the olfactory mucus.

Figure 6. Dependence of time constant for receptor current recovery on external [Na⁺]

A, mean time constants for single exponential curves fitted to receptor current recovery in solutions with modified Na⁺ concentration following IBMX exposures of 1 s (▴, as in Fig. 4) or 2 s (▿) duration. Data have been normalized for each cell to the time constant measured in normal Ringer (111 mm[Na⁺]). Data points represent the means of 3–18 cells; error bars denote standard errors of the mean. B, rate constants, calculated as the reciprocal of the mean time constants for the 1 s IBMX exposures in A, normalized to the rate constant in normal Ringer solution, which was measured in each cell. Fitted curve represents the Hill equation with K_d= 62.3 mm and n = 3.24, with minimum rate constrained to zero and a maximum normalized rate of 1.15, fitted by a weighted Marquardt–Levenberg least squares algorithm.

Figure 4. The time constant for receptor current recovery upon the return to Ringer solution is little affected by the duration of prior inhibition of Na⁺–Ca²⁺ exchange

Collected data from experiments in which the cilia of an isolated olfactory receptor were exposed, following IBMX stimulation, to reduced Na⁺ concentration (as in Fig. 2; mean of 19–20 cells) or Ni²⁺ (as in Figure 3; mean of 10–11 cells) for the durations indicated before the return to Ringer solution. In each case time constants have been normalized for each cell to the value obtained upon the immediate return to Ringer solution. Error bars represent standard errors of the mean.

Figure 2. The time constant for receptor current recovery reflects Ca²⁺ extrusion by Na⁺–Ca²⁺ exchange

Suction pipette recordings from an isolated olfactory receptor cell in response to 1 s stimulation with Ringer solution containing 500 μm IBMX. Same cell as Figure 1. Following IBMX stimulation the cilia were returned to Ringer solution either immediately (A) or following exposure for 1 s (B), 2 s (C), or 3 s (D) to a solution in which the Na⁺ concentration had been reduced to 33 mm (low Na⁺) by equimolar replacement with guanidinium. In each case the rapid decay of receptor current upon the return to Ringer solution has been fitted with a single exponential of time constant A: 0.521 s; B: 0.692 s; C: 0.561 s; D: 0.498 s. Interrupted curves in B–D reproduce the exponential fit to A for comparison. Each trace is the average of 2 or 3 trials and has been junction corrected.

Figure 1. Procedure for junction current subtraction

Suction pipette recordings from an isolated olfactory receptor cell in response to 1 s stimulation with Ringer solution containing 500 μm IBMX. Following IBMX stimulation the cilia were returned to Ringer solution either immediately (A) or after 1 s exposure to a solution in which the Na⁺ concentration had been reduced to 33 mm (low Na⁺) by equimolar substitution with guanidinium (B). Each trace is the average of 2 or 3 trials.

Figure 3. Receptor current recovery is retarded when Na⁺–Ca²⁺ exchange is inhibited by Ni02+

Suction pipette recordings from an isolated olfactory receptor cell in response to 1 s stimulation with Ringer solution containing 500 μm IBMX. Following IBMX stimulation the cilia were returned to Ringer either immediately (A) or following exposure for 2 s (B) or 3 s (C) to a solution which included 5 mm ${\text{Ni}}_0^{\text{2+}}$. In each case the rapid decay of receptor current upon the return to Ringer solution has been fitted with a single exponential of time constant A: 0.434 s; B: 0.335 s; C: 0.454 s. Interrupted curves in B and C reproduce the exponential fit to A for comparison. Each trace is the average of 2–3 trials and has been junction corrected.

Figure 5. Effect of external Na⁺ concentration on receptor current recovery

A, suction pipette recordings from an isolated olfactory receptor cell in response to 1 s stimulation with Ringer solution containing 100 μm IBMX. Following IBMX stimulation the cilia were returned either to Ringer solution, or to partially guanidinium-substituted solutions of reduced Na⁺ concentration as indicated beside each trace. B, final decay of receptor currents in low-Na⁺ solution from A, normalized to the value just following the solution change. Individual traces have been zeroed as in Fig. 5A to the initial baseline before the IBMX stimulus; note slight baseline drift. In each case the decay of receptor current has been fitted with a single exponential of time constant 99 mm: 0.067 s; 66 mm: 0.125 s; 33 mm: 0.285 s; 11 mm: 1.46 s. Time constant in normal Ringer solution 0.084 s. Each trace is the average of 2–3 trials and has been junction corrected.