SUMMARY1. The whole-cell patch-clamp technique was used to investigate the characteristics of two types of sodium current (INa) recorded at room temperature from small diameter (13-25 /Lm) dorsal root ganglion (DRG) cells, isolated from adult rats and maintained overnight in culture.2. Sodium currents were isolated pharmacologically. Internal Cs' and external tetraethylammonium (TEA) ions were used to suppress potassium currents. A combination of internal EGTA, internal F-, a low (10 gbM) concentration of external Ca2' and a relatively high (5 mM) concentration of internal and external Mg2+ was used to block calcium channels. The remaining voltage-dependent currents reversed direction at the calculated sodium equilibrium potential. Both the reversal potential and magnitude of the currents exhibited the expected dependence on the external sodium concentration.3. INa subtypes were characterized initially in terms of their sensitivity to tetrodotoxin (TTX). TTX-sensitive (TTX,) currents were at least 97 % suppressed by 041 /LM TTX. TTX-resistant (TTXr) INa were recorded in the presence of 0 3 ftM TTX and appeared to be reduced in amplitude by less than 50% in 75 /tM TTX (n= 1).4. As in earlier studies, the peak of the current-voltage relationship, the mid-point of the normalized conductance curve and the potential (Vh) at which the steady-state inactivation parameter (hoo) was 0-5 were found to be significantly more depolarized for the TTXrINa (by ca 10, 14 and 37 mV respectively). There was little difference in the slope at the mid-point of the normalized conductance curves (the mean slope factors were 5-1 mV for the TTX, INa and 4-9 mV for the TTXr current) but the h., curves for TTXr currents were significantly steeper than those for TTXS currents (mean slope factors of 3-8 and 11-5 mV respectively). Both the time to peak and the decay time constant of the peak current recorded from a holding potential of -67 mV were more than a factor of three slower for the TTXr INa than for the TTXS current.5. However, in direct contrast to the difference in activation and decay kinetics, 'slow' TTX. INa recovered from inactivation at -67 mV, or reprimed, more than a factor of ten faster than 'fast' TTXS INa. 7. The possible fundamental importance to DRG cells of such large differences in the voltage dependence of the inactivation systems (both resting inactivation and repriming kinetics) of the two sodium channel subtypes will be discussed.
Sensory neurones with their cell bodies in dorsal root ganglia (DRG neurones) transmit various types of sensory information from the periphery to the spinal cord. DRG neurones are a physically and functionally heterogeneous population, with cell bodies in adult rats ranging in size from less than 20 to over 50 ìm diameter. During the last 20 years there has been increasing acceptance that cells of different size and sensory modality also express different mixes of voltage-gated ion channels, including pharmacologically and electrophysiologically distinct subtypes of voltage-gated Na¤ channels. The initial DRG Na¤ channel classification related to the effects of the Na¤ channel blocker tetrodotoxin (TTX) and included TTX-sensitive (TTX-S) and TTXresistant (TTX-R) currents (Kostyuk et al. 1981). The first TTX-R currents to be described were slower to activate and inactivate than their TTX-S counterparts and had higher voltage thresholds for both activation and inactivation (Kostyuk et al. 1981;Schwartz et al. 1990; Journal of Physiology (1998) 1. Whole-cell and single-channel Na¤ currents were recorded from small (ca. 20 ìm diameter) cells isolated from adult rat dorsal root ganglia (DRG). Currents were classified by their sensitivity to 0·3 ìÒ tetrodotoxin (TTX), electrophysiological properties and single-channel amplitude. Cells were classified according to the types of current recorded from them. 2. Type A cells expressed essentially pure TTX-sensitive (TTX-S) currents. Availability experiments with prepulse durations between 50 ms and 1 s gave a half-available voltage (Vh) of around −65 mV but the availability curves often had a complex shape, consistent with multiple inactivation processes. Measured inactivation time constants ranged from less than 1 ms to over 100 s, depending on the protocol used. 3. Cell types B and C each had, in addition to TTX-S currents, substantial and different TTXresistant (TTX-R) currents that we have designated TTX-R1 and TTX-R2, respectively. TTX-R1 currents had a 1 s Vh of −29 mV, showed little 1 Hz use dependence at −67 mV and recovered from the inactivation induced by a 60 ms depolarizing pulse with time constants of 1·6 ms (91%) and 908 ms. They also exhibited slow inactivation processes with component time constants around 10 and 100 s. TTX-R2 currents activated and inactivated at more negative potentials (1 s Vh = −46 mV), showed substantial 1 Hz use dependence and had inactivation (60 ms pulse) recovery time constants at −67 mV of 3·3 ms (58%) and 902 ms. 4. Type D cells had little or no current in 0·3 ìÒ TTX at a holding potential of −67 mV.Current amplitude increased on changing the holding potential to −107 mV. Type D cell currents had more hyperpolarized availability and I-V curves than even TTX-R2 currents and suggest the existence of TTX-R3 channels. 5. In outside-out patches with 250 mÒ external NaCl, the single-channel conductance (ã) of TTX-S channels was 19·5 pS and the potential for half-maximal activation (Va) was −45 mV. One population of TTX-R channels had...
Members of the rat brain Kv1 family of cloned potassium channels are structurally highly homologous, but have diverse conductance and pharmacological characteristics. Here we present data on the effects of mutating residues K533 in the P-region and H471 in the S4-S5 linker of Kv1.4 to their equivalent residues in Kv1.1 and Kv1.6 on single-channel conductance and sensitivity to external tetraethylammonium cations (TEA+) and internal Mg2+. Exchange of residue K533 for its equivalent residue (Y) in Kv1.1 and Kv1.6 increased the single-channel conductance at both negative and positive potentials. This mutation is known to reduce the IC50 for external TEA+ from > 100 mM to 0.6 mM, almost identical to that for Kv1.1 (0.53 mM). We have now found that the additional exchange of residue H471 for the equivalent residue (K) in Kv1.6 increased the IC50 for external TEA+ from 0.6 mM (Kv1.4K533Y) to 2.39 mM; this is very close to that for wild-type Kv1.6 channels (2.84 mM). The mutation H471K alone was ineffective. We thus provide evidence that the S4-S5 linker does contribute to the channel's inner-pore region. Data on the block of Kv1 channels by internal Mg2+ indicate that while the binding site is probably situated within the deep-pore region, its exact location may be channel specific.
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