TEA- and apamin-resistant K(Ca) channels in guinea-pig myenteric neurons: slow AHP channels

Abstract

The patch-clamp technique was used to record from intact ganglia of the guinea-pig duodenum in order to characterize the K(+) channels that underlie the slow afterhyperpolarization (slow AHP) of myenteric neurons. Cell-attached patch recordings from slow AHP-generating (AH) neurons revealed an increased open probability (P(o)) of TEA-resistant K(+) channels following action potentials. The P(o) increased from < 0.06 before action potentials to 0.33 in the 2 s following action potential firing. The ensemble averaged current had a similar time course to the current underlying the slow AHP. TEA- and apamin-resistant Ca(2+)-activated K(+) (K(Ca)) channels were present in inside-out patches excised from AH neurons. The P(o) of these channels increased from < 0.03 to approximately 0.5 upon increasing cytoplasmic [Ca(2+)] from < 10 nM to either 500 nM or 10 microM. P(o) was insensitive to changes in transpatch potential. The unitary conductance of these TEA- and apamin-resistant K(Ca) channels measured approximately 60 pS under symmetric K(+) concentrations between -60 mV and +60 mV, but decreased outside this range. Under asymmetrical [K(+)], the open channel current showed outward rectification and had a limiting slope conductance of about 40 pS. Activation of the TEA- and apamin-resistant K(Ca) channels by internal Ca(2+) in excised patches was not reversed by washing out the Ca(2+)-containing solution and replacing it with nominally Ca(2+)-free physiological solution. Kinetic analysis of the single channel recordings of the TEA- and apamin-resistant K(Ca) channels was consistent with their having a single open state of about 2 ms (open dwell time distribution was fitted with a single exponential) and at least two closed states (two exponential functions were required to adequately fit the closed dwell time distribution). The Ca(2+) dependence of the activation of TEA- and apamin-resistant K(Ca) channels resides in the long-lived closed state which decreased from > 100 ms in the absence of Ca(2+) to about 7 ms in the presence of submicromolar cytoplasmic Ca(2+). The Ca(2+)-insensitive closed dwell time had a time constant of about 1 ms. We propose that these small-to-intermediate conductance TEA- and apamin-resistant Ca(2+)-activated K(+) channels are the channels that are primarily responsible for the slow AHP in myenteric AH neurons.

Figure 1. Afterhyperpolarization (AHP) channels in a cell-attached patch recording from an electrophysiologically identified AH neuron

Aa, the pipette potential was held at −60 mV and then stepped to −50 mV. Few channel openings were observed in the patch in the absence of action potential firing: c, closed state; o1, one channel open. b, stepping the pipette potential (V_p) from −60 mV to +60 mV for 110 ms evoked a single action current (arrow) in the neuron and this was followed by the opening of up to three channels at V_P = −50 mV. Ba and b, all-points histograms of the corresponding traces shown in A. Note the increase in P_o following AP firing. Unitary current amplitude was 0.37. c, i-V relationship of K_AHP channels at three values of V_p. A linear regression line through the data points gave a slope conductance of approximately 40 pS. C, whole-cell current clamp recording from the same neuron which generated a slow AHP following a three-pulse stimulus train. The bath contained Krebs solution and the pipette was filled with 145 mm KCl, containing 50 nm Ca²⁺.

Figure 2. Cell-attached patch recording from an AH neuron with BK and SK channels blocked with TEA (5 mm) and apamin (0.5 μm)

Aa, in the absence of APs, few channel openings were evident (the asterisk marks the opening of one channel). V_P = −20 mV. Traces b-g were recorded following 110 ms step depolarizations of the patch (V_P = +60 mV) to trigger an AP in the neuron (not shown). Following AP firing, there is an increase in channel activity in the patch. In traces d and e, channel openings continued past the end of the recording. Up to four TEA- and apamin-resistant channels are seen to open simultaneously. h, ensemble current averaged from 10 traces containing channel openings. The patch current during the step depolarization has been removed for clarity. Note slow decay of current. B, all-points histogram constructed from traces containing channel openings, revealing three equidistant peaks and fitted with Gaussian curves (closed state on left and two open states; the third open state is within the baseline). Unitary current was 0.67 pA. C, whole-cell recording after patch rupture shows prominent slow AHP following three evoked action potentials (filled bar) recorded under current clamp.

Figure 3. Outside-out patch recording from AH neurons showing two populations of K_Ca channels

The bath was perfused with Krebs solution and the patch pipette was filled with 145 mm KCl containing 500 nm Ca²⁺. A, the outside-out patch was ramp-depolarized from −100 mV to +50 mV over 10 s (upper trace). Note activation of BK-type channels (three channels open simultaneously) at potentials positive to approximately +20 mV in the current recording (lower trace). B, ramp current elicited after TEA (5 mm) was added to the Krebs solution. Openings of BK-type channels were abolished, but openings of the smaller conductance channels persisted. C, following the addition of Ba²⁺ (1 mm) to the TEA-containing Krebs solution, the opening of the smaller conductance channels was markedly inhibited at potentials negative to 0 mV.

Figure 4. Activation by cytoplasmic Ca²⁺ of TEA- and apamin-resistant K_Ca channels in an inside-out patch

Aa, two TEA- and apamin-resistant K_Ca channels were stimulated to open following wash-in of 500 nm Ca²⁺-containing high-K⁺ PS. The patch was held at 0 mV under asymmetric K⁺ concentrations at room temperature (5 mm potassium gluconate in pipette; 145 mm potassium gluconate in bath). The pipette solution contained 5 mm TEA and 0.5 μm apamin. b, time course of activation of K_Ca channels by Ca²⁺ expressed as NP_o (where N is number of channels), showing persistent activity after wash-out of 500 nm Ca²⁺ PS. The dotted lines represent gaps in continuous recordings where recordings at other potentials were executed. B, activity of K_Ca channels before (a) and in the presence of (b) Ca²⁺ PS. c, activity of K_Ca channels persisted following 5 min wash-out of Ca²⁺ PS. d, wash-in of 0 Ca²⁺ PS containing 145 mm sodium gluconate blocked current through K_Ca channels.

Figure 5. Rapid activation and deactivation of BK-type channels by Ca²⁺-PS in another inside-out patch

A, the patch which contained a single BK-type channel was held at 0 mV under asymmetrical transmembrane K⁺ concentrations. Upon wash-in of Ca²⁺-containing (10 μm) high-K⁺ PS, there was a rapid increase in the P_o of the BK channel; activity quickly reversed following wash-in of 0 Ca²⁺ PS. B, recordings from the same patch in response to ramp depolarization in the presence of 0 Ca²⁺ PS in the bath (a) and after wash-in of 500 nm Ca²⁺(b). PS ramp depolarization activated BK channels at potentials positive to about +20 mV. c, in the presence of 10 μm Ca²⁺-containing PS in the bath, the ramp depolarization activated BK channels at potentials positive to about −80 mV. TEA was omitted from the pipette solution.

Figure 6. The effect of ramp depolarization of an inside-out patch containing TEA- and apamin-resistant K_Ca channels

A, ramp depolarization from −100 to +50 mV over 10 s failed to activate channel openings except at very positive potentials (cytoplasmic [Ca²⁺] < 10 nm). B, following equilibration in 500 nm Ca²⁺-containing PS on the cytoplasmic side of the patch, ramp depolarization of the patch activated TEA- and apamin-resistant K_Ca channels throughout the ramp. Insets in each panel show expanded portions of the ramp currents around 0 mV (continuous line indicates closed channel level).

Figure 7. Lack of voltage dependence of opening of TEA- and apamin-resistant K_Ca channels

A, inside-out patch containing an estimated two channels which were activated with 500 nm Ca²⁺ high-K⁺ PS. Steady-state activity is shown at three potentials. The asterisk in trace b indicates the possible simultaneous opening of two channels. o, open state; c, closed state. B, all-points histograms of 20 s recordings of channel activity at the three potentials, fitted with superimposed Gaussian curves. Note that the curves representing the open state distribution are of roughly equal relative area. C, P_o-V plot constructed from the mean data, from recordings obtained under asymmetrical K⁺ concentrations. Data points have been fitted with a Boltzmann function, of the form: P_o = P_o,max/[1 + exp(V_0.5/V_slope)] + P_o,basal, where V_slope = 40 mV, V_0.5 is −20 mV, P_o,max was 0.56 and P_o,basal was 0.37. D, i-V relationship of TEA- and apamin-resistant K_Ca channels under asymmetric K⁺ concentrations. Data points have been fitted with a form of the GHK current equation, showing open channel outward current rectification. From the fitted curve, the limiting slope conductance at positive potentials is approximately 40 pS and the chord conductance at −50 mV is 12 pS.

Figure 8. Ca²⁺-dependent activity of TEA- and apamin-resistant K_Ca channels under symmetrical K⁺ concentrations

A, wash-in of 5 μm Ca²⁺-containing PS on an inside-out patch held at −60 mV and containing a single TEA- and apamin-resistant K_Ca channel, resulted in an increase in channel activity. B, all-points histogram plots of channel activity recorded in 0 Ca²⁺ PS and in the presence of 5 μm Ca²⁺, at −60 mV, fitted with superimposed Gaussian curves. Note basal activity of K_Ca channels (left graph). C, P_o-V curve constructed from the mean data collected from three to eight patches exposed to 2–5 μm cytoplasmic Ca²⁺. Data points have been fitted with a straight line indicating no voltage dependence. D, i-V plot of the mean unitary current ± s.e.m., fitted with a linear regression line between −60 and +60 mV, yielding a unitary conductance of 62 pS.

Figure 9. Kinetic analysis of TEA- and apamin-resistant K_Ca channel activity recorded under asymmetrical K⁺ concentrations

Aa, activity in a typical inside-out patch at 0 mV in 0 Ca²⁺ PS; b, steady-state activity following wash-in of high-Ca²⁺ PS on the cytoplasmic surface. The trace was digitally filtered at 1 kHz and contains two channels, but simultaneous openings were rare (c). Amplitude histogram plot of the idealized transitions to the open state (o) and to the closed state (c), each fitted with a Gaussian curve. The single channel current was 1.07 and the P_o was 0.39. B, dwell time distributions showing that the open dwell time was fitted with a single exponential curve with a time constant of 1.25 ms (a), while the closed dwell time was best fitted with the sum of two exponential functions having time constants of 1.44 and 7.89 ms (b).