Potassium currents in freshly dissociated uterine myocytes from nonpregnant and late-pregnant rats

Abstract

In freshly dissociated uterine myocytes, the outward current is carried by K+ through channels highly selective for K+. Typically, nonpregnant myocytes have rather noisy K+ currents; half of them also have a fast-inactivating transient outward current (ITO). In contrast, the current records are not noisy in late pregnant myocytes, and ITO densities are low. The whole-cell IK of nonpregnant myocytes respond strongly to changes in [Ca2+]o or changes in [Ca2+]i caused by photolysis of caged Ca2+ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca2+ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at -80, -40, or 0 mV and digital subtractions, the whole-cell IK of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately -61 and -22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca2+-activated K+ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca2+-sensitive K+ channels also have a half-open probability at 68 mV, and are less sensitive to Ca2+ than similar channels in taenia coli myocytes. Ca2+-activated K+ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30-35% of the total IK in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute approximately 20% of total IK in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K+ currents that include a suppression of the ITO, a redistribution of IK expression from large-conductance Ca2+-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca2+ sensitivity, and a positive shift of the activation of some large-conductance Ca2+-activated channels.

Figure 1

Outward currents of typical nonpregnant and late-pregnant rat uterine myocytes. Currents at HP −80 and −50 mV are shown at same scales for comparison (depolarized for 258 ms in 10-mV increments to +70 mV). Symbols above top current traces indicate where current was measured for i-V curves. (A–C) Nonpregnant myocytes, 16.8 pF. Current has frequent and large fluctuations (noisy). (A) HP −80 mV. I_TO is clearly visible, peaking at ∼3.5 ms, declining rapidly to a more gradual current that reached maximum at 33 ms. From this maximum, decay is faster than in late-pregnant myocytes (D). Also note greater outward rectification. (B) Same myocyte, HP −50 mV. I_TO is now absent (see inactivation relation in Fig. 6 A). Maximum current is 41% of that at HP −80 mV. Current noise remains about the same; decay is less. (C) i-V relations. Note the differences between maximum current and end-of-pulse current, indicating degree of decay; also obvious outward rectification. (D–F) Myocyte from 19-d pregnant uterus; cell capacitance 108 pF. (D) HP −80 mV. Currents develop gradually, reaching a maximum at ∼35 ms. Currents are generally smooth, with little noise fluctuations. They are also well-sustained over several hundred milliseconds. More decay is evident over several seconds. First current appeared at approximately −40 mV. Some outward rectification is evident to 0 mV; thereafter, rectification is slight. In traces from −20 to 20 mV, a small early distortion may be the transient outward current. (E) Same myocyte at HP −40 mV. Total current is now 17% that at HP −80 mV. No decay is evident. (F) i-V relations of currents at maximum and at end. Note that outward rectification is very slight, as is decay.

Figure 6

Steady state activation and inactivation properties of component currents of nonpregnant and late-pregnant uterine myocytes. (A) Properties of I_TO in nonpregnant myocytes. For V-h relation (left curve and ordinate ; data from six myocytes shown as means ± SEM, if scatter is larger than symbol), two-pulse protocol similar to those for Fig. 5 was used. Solid curve is Boltzmann distribution function with half inactivation at −76.5 mV and a slope of 6.9 mV. At −50 mV, the relative current is 0.01; at −40 mV, 0.001. For V-g relation (right curve and ordinate ; eight myocytes), relative conductance as a function of maximum conductance was obtained for each myocyte as the asymptotic value at 120 mV. Half activation is at +5 mV with a slope factor of 24.3 mV. (B–D) Properties of component currents. See test for paradigm of extracting C₁, C₂, and C₃ currents. In these panels, data from nonpregnant myocytes are represented by hollow symbols and their Boltzmann distribution by solid lines. Data from late-pregnant myocytes are represented by filled symbols, and their Boltzmann distribution functions by broken lines. In B and C, V-h relations are rescaled from Fig. 5. (B) Properties of C₁ currents. For nonpregnant myocytes (data from seven myocytes which had no I_TO), half activation is at 7.2 mV with a slope factor of 24.6 mV. For late-pregnant myocytes (data from 22 myocytes), half activation is at 7.7 mV with a slope factor of 23.7 mV. “Window current” is present in both types of myocytes. (C) Properties of C₂ currents. For nonpregnant state (seven myocytes), half activation is at 3.9 mV with a slope factor of 17.7 mV. For late-pregnant state (11 myocytes), half activation is at 4.2 mV with a slope of 22.1 mV. Window currents are larger than those in C₁ currents. (D) Properties of C₃ currents that do not inactivate. For nonpregnant state (seven myocytes), half activation is at 39.1 mV, slope 17.7 mV. For late-pregnant state (12 myocytes), half activation is at 63.4 mV, slope 16.7 mV. Half-activation voltages are significantly different (see text for details).

Figure 5

Voltage–steady state inactivation relation of outward current in nonpregnant and late-pregnant myocytes. Two-step protocol, holding potential −80 mV, conditioning step 10 s duration, test step 180 ms. Current of test step in presence and absence of the conditioning step is plotted on ordinate as relative current. Conditioning voltage on abscissa. Symbols are data (means ± SEM). (A) Nonpregnant myocytes. Data from eight myocytes (9.2, 18, 9.2, 11, 15.4, 25, 13.6, and 16.4 pF). The complex relation requires three components for fitting. C₁, comprising 59% of total K⁺ current, is half inactivated at −59.5 mV, with a slope factor of 13.4 mV. C₂, comprising 30%, is half inactivated at −22.9 mV with a slope of 4.1 mV. C₃, comprising 11%, does not inactivate. (B) Late-pregnant myocytes. Data from seven myocytes; three from 17-d pregnant uteri (89, 58, and 82.4 pF); two from 18-d pregnant uteri (80 and 96 pF), and two from 19-d pregnant uteri (93 and 180.4 pF). The complex relation also requires three components: C₁, comprising 67% of total I_K, is half inactivated at −62.7 mV, with a slope factor of 6.3 mV; C₂, comprising 23%, is half inactivated at −21.2 mV, with a slope factor of 5.7 mV; and C₃, comprising 10%, does not inactivate. Although the half-inactivation voltages and slope factors are similar to those of nonpregnant myocytes, C₁ has enlarged at the expense of C₂.

Figure 2

Effects of [Ca²⁺]_o on outward currents of nonpregnant and late-pregnant rat uterine myocytes. (A and B) Nonpregnant rat in estrus; cell capacitance 5.0 pF. (A) Holding potential −80 mV, depolarized for 245 ms in 10-mV increments to 70 mV. (A₁) In 1 mM Ca²⁺. Note presence of a I_TO. (A₂) In 30 mM Ca²⁺. Little effect, possibly because all currents are fully expressed. (A₃) Difference between currents in 30 and 1 mM Ca²⁺ verify the general lack of effects of increasing [Ca²⁺]_o. (B) HP −50 mV; same voltage protocol. (B₁) In 1 mM Ca²⁺, I_TO is inactivated; current rises gradually and is maintained for 245 ms with little decay. (B₂) 30 mM Ca²⁺. Marked increase in outward current, mostly in I_TO, but also some in steady state current, as is evident in difference current (B₃). (C and D) Late-pregnant myocytes; from 17-d pregnant uterus (C), cell capacitance 106 pF; from 18-d pregnant uterus (D), cell capacitance 102 pF. Cells were held at −60 mV, and depolarized by 150-ms steps in 10-mV increments to 40 mV. (C_1–3) Effects of lowering [Ca²⁺]_o from 3 to 0 mM. (C₁) In 3 mM Ca²⁺, inward I_Ca was present and I_K was well maintained. (C₂) In 0 mM Ca²⁺, I_Ca disappeared but I_K is essentially unchanged. (C₃) Difference current between C₂ and C₁. Note that difference current represents the inward I_Ca, with little difference in I_K. (D_1–3) Effects of raising [Ca²⁺]_o from 3 to 30 mM. Conventions similar to those in C_1–3. (D₁) In 3 mM Ca²⁺, I_Ca and I_K serve as bases for comparison. (D₂) In 30 mM Ca²⁺, I_Ca increased appreciably, but I_K remained essentially unchanged. (D₃) Difference current shows changes in I_Ca, but little change in I_K.

Figure 3

Responses of small multicellular preparations of late-pregnant myometrium to conditions that affect inward I_Ca. Double sucrose-gap method. (A) Effects of Mn²⁺ (5 mM). Preparation from 19-d pregnant uterus. Total “nodal” capacitance, 0.1 μF. (A₁) Action potential elicited by constant current step. (A₂) Action potential after 5 min of superfusion with Krebs solution containing Mn²⁺, showing blockade. (A_3–6) Superimposed composite currents under voltage-clamp conditions. Numbers at left margin of each trace represent command voltage step. Traces marked 1 are from control conditions, traces marked 2 are from after treatment with Mn²⁺. Note that whereas Mn²⁺ effectively blocked inward I_Ca, it produced no detectable changes in steady state I_K. (B) Effects of increasing [Ca²⁺]_o from 1.9 to 8 mM. Preparation from 20-d pregnant uterus. Total nodal capacitance, 0.07 μF. (B₁) Action potential elicited by constant current in 1.9 mM Ca²⁺. (B₂) Action potential in 8 mM Ca²⁺ shows a faster rate of rise and a higher amplitude, consistent with increased I_Ca. (B_3–6) Superimposed composite currents under voltage-clamp conditions. Traces marked 1 are from 1.9 mM Ca²⁺, traces marked 2 are from 8 mM Ca²⁺. Because of limitations of method, effects of procedures on I_K can only be examined at steady state (500 ms) when inward current has inactivated. Note that, whereas increased [Ca²⁺]_o increased I_Ca and increased overlap artifact in the early part of the outward current, it did not produce significant changes of steady state I_K. The constancy of I_K in these preparations, which were not treated with enzymes and their myocyte interior was not exposed to EGTA, is consistent with similar observations made on dissociated myocytes.

Figure 4

Effects of photolysis-induced increase of [Ca²⁺]_i on nonpregnant, late-pregnant uterine myocytes and on taenia coli myocyte. Caged nitr 5–Ca complex was introduced intracellularly by diffusion from pipette (see text for details). In each panel, five consecutive traces, recurring at 3-s intervals, are shown. Traces marked C represent three superimposed traces of depolarization-induced whole-cell I_K in cells that have been loaded with nitr 5–Ca complex, but not irradiated. Traces marked F represent the fourth trace, during which myocyte was exposed to 360 nm light at time indicated by bar beneath traces. Traces marked F+1 represent the fifth trace in series. (A and B) Nonpregnant myocytes, 10.6 and 8.4 pF, respectively. In A, average I_K showed an increase upon irradiation. The increase in this cell is unusually large. Other changes also evident include more prominent current noise, downward shift of baseline, indicating holding current became more inward, and larger tail current. In B, increase in current noise is especially evident. (C–E) Late-pregnant myocytes; from 18-d pregnant uterus, 78 (C) and 60 (D) pF, and 19-d pregnant uterus, 120 pF (E). These examples show representative responses in late-pregnant myocytes. (C) Typical of 44% of test samples (36 myocytes), this cell showed no response. (D) In this myocyte, in addition to an increase in average I_K, there was an inward shift of holding current, an increase in tail current, and an increase of current noise. (E) In this myocyte, response consisted of an increase in average I_K and in current noise. 56% of all samples responded as in D and E. (F) Response of a representative taenia coli myocyte, which is known to have abundant maxi-K channels. Large increase in average I_K and increase in current noise occurred in 97% of 29 cells tested.

Figure 7

Effects of [Ca²⁺]_o on activation of component currents of whole-cell I_K in nonpregnant and late-pregnant uterine myocytes. Symbols represent means ± SEM of five nonpregnant (A and B) and nine late-pregnant myocytes (C and D). Solid lines represent Boltzmann distributions. In nonpregnant but not late-pregnant myocytes, 30 mM Ca²⁺ caused a positive shift of V-g relation.

Figure 8

Effects of tetraethylammonium chloride on component currents of I_K of late-pregnant uterine myocytes. (A–C) TEA, 0.5 mM. (D) TEA, 2 mM. In A and B, traces of residual current in TEA (I_TEA, light traces) are overlaid on traces of current before TEA (I_control, heavy traces) at same voltages. For clarity, only selected traces are shown. (A) Myocyte from 20-d pregnant uterus, 191 pF. Traces shown are I_LP1's, which are difference currents between those obtained at HP −90 and −40 mV. (B) Traces shown are I_LP2,3, obtained directly by recording at HP −40 mV. TEA reduction of average current is associated with marked reduction of peak-to-peak current fluctuations. The y-axis labels (0, 30, and 60 mV) identify I_control current records of I_LP2,3(heavy traces), and 0.5 mM TEA causes reductions in the current at each voltage step (light traces, below). At faster time scales (not shown), TEA does not affect activation kinetics (for 60-mV step, τ_control = 13 ms, τ_TEA = 15 ms). (C) Difference currents, I_control − I_TEA at all voltage steps for myocytes in A, representing currents blocked by TEA (5.7 pA/pF at 60 mV), which does not decay over 2.1 s. Calibrations are the same as in A. (D) TEA, 2 mM. Myocyte from 17-d pregnant uterus; 126.6 pF. Traces are difference currents, I_control − I_TEA, at all voltages. Although it caused a greater block (12.6 pA/pF at 60 mV, 2.1 s) than other higher concentrations, their effects involve also some decaying component, making them less useful for differentiating channel types.

Figure 9

Effects of charybdotoxin (100 nM) on I_K of uterine myocytes. Conventions are similar to those in Fig. 8, except that A and E represent directly recorded total currents; I_ChTX (light traces) overlaid on I_control (heavy traces). (C, D, G, and H) are different currents, or currents blocked by ChTX. (A–D) Nonpregnant myocyte. 18.4 pF. ChTX reduced peak-to-peak current fluctuations. In A, I_TO is distinct in traces of −10, 10, and 30 mV in I_control, and is blocked by ChTX. In B, at HP −50 mV, only I_NP2,3 is elicited. Effects of ChTX are rather small, and are not manifested until more positive than 50 mV. (C) Difference currents, I_control − I_ChTX at HP −80 mV. For clarity, only two traces at fast time scale are shown. Note the particularly prominent block on the I_TO, manifested here as an initial surge, peaking at 3 ms. The subsequent current seen in the 70-mV trace is clearly of a different and noisy type. In the full trace (not shown), the blocked current shows no decay. (D) Difference currents, I_control − I_ChTX at HP −50 mV confirm that ChTX had no effect until beyond 50 mV. (E–H) Myocyte from 20-d pregnant uterus; 117.6 pF. At HP −80 (E) and −50 (F) mV, I_ChTX for the −10-mV trace is superimposed on I_control. (G) Difference currents, I_control − I_ChTX for HP −80 mV, on a fast time scale. The blocked currents show an initial hump, contrast with the blocked I_TO in C, followed by another sustained current. (H) Difference currents, I_control − I_ChTX at HP −50 mV.

Figure 10

Effects of iberiotoxin (1 nM) on I_K of uterine myocytes. Selected traces for clarity. I_IbTX traces are lighter and overlaid on I_control traces from the same voltages. (A–D) Nonpregnant myocyte with I_TO; 18 pF. (A) HP −80 mV, showing total I_K. (B) HP −40 mV, showing I_NP2,3 for same voltage steps as in A. (C) Difference currents, I_control − I_IbTX at HP −80 mV, (C₁) at a fast time scale to show that initial part of the blocked current coincided with I_TO. (D) Difference currents at HP −40 mV. (E–G) Nonpregnant myocyte without I_TO; 18 pF. (E) HP −80 mV. (F) HP −40 mV. Traces are of same voltages as in E. (G) HP 0 mV, showing I_NP3. IbTX reduces peak-to-peak fluctuations and average currents, most notably in I_NP3, but also evident in directly recorded currents (A, B, E, and F), and as difference currents (C and D). Other features of note: (a) IbTX effect is not evident until V > 20 mV (A and E), probably because susceptible current is not activated; (b) IbTX blocks a part of the I_TO at all voltages (A and C₁), but the blocked current is different from that blocked by ChTX (Fig. 9), suggesting that I_TO is not a homogenous current; (c) unlike most maxi-K currents shown, some IbTX-susceptible currents show an appreciable rate of decay (C₂). (H–K) Late-pregnant myocytes. (H) Myocyte from an 18-d pregnant uterus, 105.4 pF. HP −90 mV. This myocyte has very little IbTX-susceptible currents, as can also be seen in difference currents in J. (I) Myocyte from 18-d pregnant uterus, 137 pF. Main responses are similar to those described for nonpregnant myocyte. Difference currents are shown in K.

Figure 11

Effects of 4-aminopyridine on I_K of uterine myocytes. Selected traces for clarity. I_4-AP (light traces) overlaid on I_control of same voltage steps. (A–D) Nonpregnant myocyte with I_TO, 15.6 pF. 4-AP, 5 mM. While 4-AP markedly reduced total I_K (A) and I_NP2,3 (B), it did not block the I_TO (A), as also shown in difference currents in C. It also did not reduce current fluctuations in direct recording (B), or in difference currents (D). These effects are consistent with 4-AP actions on K_v channels. (E–G) Myocyte from 18-d pregnant uterus. 162 pF. 4-AP, 1 mM. (E) HP −90 mV, showing total I_K. Note that current at 2.1 s (end of step) is slightly more depressed by 4-AP than current at 35 ms (maximum), a feature also seen in dose–response relations in H. (F    and G) HP −40 mV, showing I_LP2,3. Noisy current is obvious at +60 mV, but 4-AP has no effect on peak-to-peak fluctuations. Slowing of the rate of activation by 4-AP is already evident, but more so at a faster time scale in G. At 60 mV, τ_control = 36 ms, τ_4-AP = 64 ms. This effect on kinetics could cause the wrong conclusion that 4-AP is selective for some transient current. Comparing E and F, and also A and B, it is clear that main effects of 4-AP are exerted on the C₁ components (I_LP1 and I_NP1). (H) Dose–response relation of 4-AP on currents at 35 ms (I_max; hollow symbols) and at 2.1 s (filled symbols). Hill plot, abscissa, log concentration; ordinate, (1 − P)/P where P is I_4-AP/I_cont. ED₅₀ is at 1 − P/P = 1. I_K at 2.1 s is almost three times more susceptible than I_max.

Figure 12

Effects of dendrotoxin (200 nM) on I_K of uterine myocytes. In all panels, I_DTX (light trace) is overlaid on I_control. For clarity, only selected traces are shown. (A and B) Nonpregnant myocyte, 19.2 pF. (A) HP −80 mV, eliciting total I_K. (B) HP −40 mV, eliciting I_NP2,3. DTX has no effect on this myocyte. (C and D) Myocyte from 19-d pregnant uterus, 93.2 pF. (C) HP −80 mV. Total I_K is reduced slightly by DTX (I_DTX/I_cont = 0.96 at maximum current and 0.97 at end). (D) HP −40 mV. DTX effect on I_LP2,3 is appreciable (I_DTX/I_cont = 0.54 at maximum and at end). Note that in DTX, current fluctuations are unchanged. Effects are consistent with DTX blocking a delayed rectifier current (see text for details).

Figure 13

Relative activities of small and large-conductance K⁺ channels in membrane patch from late-pregnant uterine myocyte. (A) Detached inside-out patch from 18-d pregnant uterine myocyte. Holding potential +40 mV. Pipette solution (facing outside of membrane, mM): 135 NaCl, 5.4 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, 5 glucose. Bath solution (facing inside of membrane, mM): 135 KCl, 0.6 EGTA, 0.1 CaCl₂ (pCa = 8), 10 HEPES. This patch has both small- and large-conductance channels, infrequently encountered in uterine myocyte patches. Closed state (c) marked at left margin. Dotted lines indicate different open levels: first level is for small-conductance channel, second level is for large-conductance channel, third level is for simultaneous openings of small and large channels. Unit conductance for small channel is 41 pS; for large channel, 180 pS. (B) Activity histogram of channels in A. Abscissa in 0.1 pA bins; ordinate in log scale, total data points, each representing 150-μs duration (in a 16-s continuous recording). Peak a represents closed state, b a small-conductance channel alone, c a large-conductance channel alone, and d a small and large channel simultaneously. P _o for the large channel is 0.007 and for the small channel is 0.15 (21× higher; see text for other details).

Figure 14

Voltage–open probability relations of maxi-K channels from taenia coli myocyte and late-pregnant uterine myocyte, and effects of [Ca²⁺]_i on them. (A and B) Data from representative individual patch for illustration. (C) Summary of data. In A and B, solid curves are Boltzmann distributions: P _o = [1 + exp(V_h − V)/ k]⁻¹, where V_h is voltage at which P _o = 0.5, and k is logarithmic voltage sensitivity. Filled symbols for pCa 8; hollow symbols for pCa 7. (A) For taenia coli channel, V_h and k are, respectively, 63.2 and 7.9 mV for pCa 8, and 35.4 and 8.9 mV for pCa 7. (B) For late-pregnant myometrial channels, they are, respectively, 76.3 and 9.1 mV for pCa 8, and 60.3 and 10.4 mV for pCa 7. Differences: in myometrial channel, V_h is more positive, k is shallower, and negative shift of V_h on increasing [Ca²⁺]_i is less. (C) Average P _o-V relations of late-pregnant maxi-K channel compared with those of taenia coli channel. Curves are computed Boltzmann distributions based on mean data of V_h and k obtained individually from six taenia coli patches and nine myometrial patches. Each curve is identified by average V_h value used; triangles for myometrial channels, circles for taenia coli channels. Filled symbols for pCa 8, hollow symbols for pCa 7. See text for data.