(Received for publication, March 31, 1995; and in revised form, June 5, 1995)
From the
The auditory receptor epithelium is an excellent model system
for studying the differential expression of ion channel genes. An
inward rectifier potassium current is among those which have been
measured in only subsets of chick cochlear hair cells. We have cloned
and characterized an inward rectifier potassium channel (cIRK1) from
the chick cochlear sensory epithelium. cIRK1 functional properties are
similar to those of the native channel, and the transcript encoding
cIRK1 is limited to the low frequency half of the epithelium. This
localization is in agreement with the distribution of the native hair
cell current, suggesting that the differential current expression is
transcriptionally regulated. The primary structure of cIRK1 is highly
homologous to the mouse inward rectifier IRK1. However, we found that
cIRK1 exhibited reduced single-channel conductance (17 picosiemens) and
lower sensitivity to Ba block (K = 12
µM). We identified Gln-125 near the putative pore region
as being responsible for these differences. Site-directed mutagenesis
was used to change Gln-125 to Glu (the residue in IRK1), resulting in a
channel with a single-channel conductance of 28 picosiemens and a
Ba
block of K = 2 µM. We
propose that Gln-125 may form part of the external vestibule of the
pore.
The auditory receptor epithelium of the chick, the basilar papilla, consists of hair cells and supporting cells. Approximately 10,000 hair cells are found in the receptor epithelium(1) . These cells receive either primarily efferent or primarily afferent innervation (2) . Those cells that receive predominantly afferent innervation (tall hair cells) are the primary receptors that transduce sound(3) . These cells convert the mechanical energy of sound to a coded electrochemical one(4, 5) . Tall hair cells are arranged tonotopically along the long axis of the basilar papilla with cells toward the apex responding to low frequency sound and cells toward the base responding to high frequency sound(6) . In the chicken, the membrane properties of tall hair cells differ along the tonotopic axis, and potassium channels, in particular, play a pivotal role in determining these properties(7, 8, 55) .
Electrophysiological experiments have shown three potassium currents
in tall hair cells: a calcium-activated potassium current; a delayed
rectifier potassium current; and an inward rectifier potassium current (8, 55) . Of these, both the inward rectifier and
delayed rectifier currents have been shown to be present in hair cells
responding to low frequency sound (apical hair cells), while the
calcium-activated potassium current has been shown to be present in
those hair cells responding to high frequency sound. The inward
rectifier potassium current contributes to determining the resting
membrane potentials and the membrane response times of these cells. The
inward rectifier current present in tall hair cells has sharp inward
rectification, prolonged open times, and is sensitive to block with
external Ba and Cs
(8) .
Several inward rectifier potassium channels with differing
electrophysiological properties and mechanisms of activation have
recently been cloned from a number of tissues and species. Among these
are IRK1 from mouse macrophages(9) , ROMK1 from rat
kidney(10) , GIRK1 (11, 12) and rcK(13) from rat heart, HIRK1 from human
hippocampus(14) , and RB-IRK2 from rat brain and RBHIK1 from
rabbit heart(15, 16) . Of these, GIRK1 is activated
through a G protein, rcK
is opened by ATP, and the
remainder are activated by hyperpolarizing voltages. Apart from ROMK1,
which has been shown to have moderate rectification, the rest of these
channels have sharp inward rectification. All of these channels can be
blocked by application of external cations (Ba
and
Cs
). Hydropathy analyses of all these channels predict
two membrane-spanning hydrophobic domains (M1 and M2) in the middle of
the protein(9, 10, 11, 12) . These
membrane-spanning domains are separated by the H5 region. The H5 domain
forms an important part of the pore in potassium
channels(17, 18) . The amino acid sequences of these
inward rectifier channels are most conserved in the membrane-spanning
and H5 regions. A number of experiments have helped elucidate their
structure-function relations. Recently, it was reported that replacing
the C terminus of ROMK1 with that of IRK1 altered its
electrophysiological properties of Mg
block and
K
conductance(19) . Furthermore, mutation of a
single residue (Asp-172) in the M2 segment of IRK1 demonstrated that
this residue is important in determining inward rectification through
internal Mg
block and the rate of
activation(20, 21) .
We report here the cloning of
an inward rectifier potassium channel (cIRK1) from the chick basilar
papilla and demonstrate its localization in the apical half of the
epithelium. cIRK1 has a reduced single-channel conductance and a
decreased sensitivity to block with external Ba compared with the mouse inward rectifier, IRK1. Both differences
were removed by substituting a glutamate residue for glutamine 125 in
cIRK1. Glutamine 125 lies between the proposed M1 and H5 regions of the
channel. This residue may be part of the external vestibule of the
pore.
In experiments intended to identify the prevalence
of these transcripts in the two halves of the basilar papilla, template
cDNAs for PCR were synthesized by random priming of poly(A) RNA isolated from microdissected apical and basal halves of the
basilar papilla. The primers used, based on the sequence of cIRK1, had
the following sequences: TCTTCAGCCACAATGCCGTG and AGACCAGGAATATGCGGTC.
Equal amounts of cDNA were used in each PCR. The amounts of cDNA were
determined by colorimetry (DNA DipStick
, Invitrogen). The
PCR was carried out in a 20-µl volume with 4 mM
MgCl
, 5 µCi of [
P]dCTP, and 0.5
unit of Taq polymerase using the following conditions: 94
°C, 1 min; 45 °C, 2 min, 72 °C, 3 min for 60 cycles.
2-µl aliquots were removed every 10 cycles from the 30th cycle
onward. These aliquots were then separated on a denaturing
polyacrylamide gel (6 M urea, 6% acrylamide, 1
TBE (90
mM Tris borate, 2 mM EDTA). The amount of
radioactivity in the band of the anticipated size was determined using
a PhosphorImager. Similar experiments were performed with primers
specific to the chick calbindin-D28k sequence. The primers used had the
following sequences: CAGGGTGTCAAAATGTGTGC and GGTCAAGACGAGCCATTTCG.
They were based on the chick cDNA sequence (24) and were
chosen to span several introns(25) . The experimental
conditions were identical with those used in the experiment done to
localize cIRK1, except that the annealing temperature was raised to 55
°C and the reaction was terminated after 30 cycles.
Electrophysiological recordings
from Xenopus oocytes were done using a TEV-200
two-microelectrode voltage clamp amplifier (Dagan), to record whole
cell currents, or an Axopatch 200 patch-clamp amplifier (Axon
Instruments), to record single-channel currents(30) . For
whole-cell and cell-attached patch-clamp recording, the bath solution
contained 140 mM potassium aspartate, 10 mM KCl, 1.8
mM CaCl, 10 mM HEPES (pH 7.4; titrated
with KOH). In the cell-attached configuration, this solution clamps the
membrane potential near 0 mV. The same solution was present in the
patch pipette. Whole cell currents were low-pass-filtered at 1 kHz
using an internal 4-pole Bessel filter and digitized at 1 or 2 kHz.
Single-channel currents were low-pass-filtered at 1 or 2 kHz using an
8-pole Bessel filter and digitized at 4 or 8 kHz, respectively. Leak
currents were subtracted assuming a linear leak at positive voltages,
and capacitive currents in ramp experiments were subtracted using
templates constructed by fitting a smooth function to records with no
openings. BaCl
was applied using a gravity-driven perfusion
system. To ensure equilibrium, the recording chamber was perfused with
at least four chamber volumes (4
250 µl) at a rate of
approximately 3 ml/min. All currents were recorded at room temperature
(22-23 °C). Data acquisition and analysis were done using
pCLAMP 6.0. (Axon Instruments). For further analysis we used Sigmaplot
(Jandel). All results are reported as means ± S.D.
Figure 1:
Nucleic acid and deduced amino acid
sequences of cIRK1. The nucleic acid sequence of the longest inward
rectifier clone and its deduced amino acid sequence are shown. The
purported membrane-spanning and pore regions in the deduced amino acid
sequence are underlined. The initiator methionine was ascribed
according to the consensus Kozak sequence. Also indicated are the
possible sites for post-translational modification (MacVector 4.1,
IBI): an N-glycosylation site (#), one protein kinase C phosphorylation
site (), four protein kinase A phosphorylation sites (+), and
two tyrosine kinase phosphorylation sites (♦). Indicated below
the deduced amino acid sequence are the amino acids which differed in
IRK1 (9) . These are clustered around the region connecting the
M1 and H5 regions and near the C terminus.
Figure 3:
Size and tissue distribution of cIRK1
transcripts. Northern analysis of poly(A) RNA obtained
from brain, cerebellum, cochlea, muscle, liver, and heart. A DraI fragment (1.9 kb) of cIRK1 was used as a probe. The blot
was washed in 0.1
SSC at 65 °C. The major band of 5.4 kb (arrow) with a less intense band of 2.2 kb and several minor
bands of different sizes are present. The migration of the RNA size
markers is indicated on the left (sizes in
kilobases).
We then attempted to localize this channel within the basilar papilla. This was done by a PCR using primers specific to the cIRK1 clone. cDNAs obtained from the apical and basal halves of the basilar papilla were used as templates in two separate reactions. A PCR product of the anticipated size was seen only in the reaction which contained cDNA obtained from the apical half of the basilar papilla (Fig. 4). There was no evidence of such a product in the basal half of the basilar papilla even after 60 cycles. A parallel control experiment was done to confirm the integrity of the cDNA obtained from the two halves of the receptor epithelium. A similar PCR was performed using primers specific for calbindin D28k, a calcium binding protein abundant in the chick basilar papilla(32) . In this experiment, and in contrast to the result obtained with the cIRK1 primers, a product of the expected size was present in the PCR where cDNA from the basal half of the epithelium served as the template (Fig. 4). A much less intense product of the anticipated size was also present in the reaction in which cDNA from the apical half of the epithelium was used as the template (Fig. 4). Although we have not attempted to test the prevalence of these transcripts by competitive PCR, we are confident of these results since the difference in abundance of the calbindin transcript between the two halves of the basilar papilla was greater than two orders of magnitude (860,000 versus 3,000 units as measured by a PhosphorImager). Moreover, this ratio which was present at the 15th cycle of the PCR was maintained at the 30th cycle. Each reaction was done in triplicate with a view to controlling for differences in amplification efficiency. Furthermore, in order to control for possible variations in mRNA isolation and cDNA synthesis, these steps were repeated, and PCRs were performed on these samples with the same results (data not shown).
Figure 4: Differential distribution of cIRK1 transcripts along the tonotopic axis of the basilar papilla. A, the differential distribution of cIRK1 and calbindin transcripts across the basilar papilla (cartoon). The upper panel shows the products of RT-PCRs (in triplicate) with primers specific to cIRK1 after 30, 40, 50, and 60 cycles using cDNA obtained from the apical (left) and basal (right) halves of the basilar papilla. The product is seen only in the PCRs which had cDNA from the apical half of the basilar papilla. The lower panel shows the products from a similar experiment using primers specific to calbindin-D28k after 30 cycles. In contrast to the pattern obtained with cIRK1, the product is seen predominantly in the basal half of the epithelium. B, quantitation of PCR products from the two halves of the basilar papilla. The signals from the triplicate samples from each cycle point were measured as one value using a PhosphorImager. These values are plotted as a function of PCR cycle number for both the apical and basal halves of the basilar papilla.
Figure 5:
Electrophysiological properties of cIRK1
expressed in Xenopus oocytes. A, whole-oocyte
currents evoked by 900-ms step voltage changes between -150 and
+50 mV in 20-mV increments from a holding voltage of 0 mV.
Currents were elicited at intervals of 5 s. Data were low-pass filtered
and digitized at 1 kHz. Leak current was subtracted assuming that a
linear component of the current-voltage relation at positive voltages
was mainly due to leak. This seems a reasonable assumption because
unsubtracted macroscopic recordings from tight-seal macropatches showed
little or no current at positive potentials, and single-channel
recording showed no openings above the expected reversal potential
(-10 to 0 mV). Capacitive transients were not subtracted. B, the corresponding steady-state current-voltage relation.
Current was measured as the average of at least 10 sampling points near
the end of the pulse. C, single-channel currents from a
cell-attached patch recorded at the indicated membrane voltages. Data
were low-pass filtered at 1 kHz and digitized at 4 kHz. D,
corresponding single-channel current-voltage relation. Each point
represents the mean current amplitude of single-channel openings. Solid line is the best-fit linear regression with a slope of
16 pS. For all recordings the external solution contained 150
mM K (see ``Experimental
Procedures'').
Figure 2: Alignment of cIRK1 with representative members of the inward rectifier family in the region between M1 and H5. The Clustal method (54) was used to align the amino acid sequences. Amino acid identities with cIRK1 are shown as dashes; gaps in sequences introduced for alignment purposes are shown as blank spaces. Position 125 in cIRK1 (Q) and IRK1 (E) and corresponding positions in other sequences are shown in bold. The overall amino acid similarity between cIRK1 and the different channels are: IRK1, 93% (9) ; RB-IRK2, 69%(15) ; RBHIK1, 94%(16) ; HRK1, 58%(14) ; GIRK1/KGA, 44%(11, 12) .
Figure 6:
The effect of Q125E on single-channel
conductance of cIRK1. A, single-channel ramp current expressed
by wild-type cIRK1. The trace shown is the average of five consecutive
responses. Voltage ramp was delivered at 2.2 mV/ms. Data were filtered
at 1 kHz and digitized at 20 kHz. Solid straight line represents the best-fit linear regression with a slope of 19 pS
(calculated between -150 and -10 mV). B,
single-channel ramp current expressed by mutant cIRK1 (Q125E). Voltage
ramp was delivered at 0.44 mV/ms. Current is interrupted by a brief
complete closure of the channel. Data were filtered at 1 kHz and
digitized at 4 kHz. The estimated slope conductance was 33 pS (solid straight line). C, single-channel
current-voltage relation determined from steady-state records from
oocytes expressing wild-type (n = 3) and Q125E (n = 1). Mean current amplitude at each voltage was determined
from amplitude histograms. Standard deviation bars are not apparent
when they are not larger than the symbol. Slope conductances (solid
lines) were 16.3 and 25.6 pS for wild-type and mutant,
respectively. Estimated pooled averages from steady-state and ramp
experiments were 16.9 ± 1.7 (n = 4) and 28.3
± 3.9 (n = 4), wild-type and mutant,
respectively. All recordings from cell-attached patches with 150 mM K in the recording
pipette.
Figure 7:
The
effect of Q125E on the sensitivity of cIRK1 to Ba block. A and C, whole-oocyte cIRK1 currents in
the absence (control) and presence of 5 µM BaCl
from oocytes expressing wild-type and mutant channels,
respectively. Currents were recorded and analyzed as indicated in the
legend to Fig. 5. B and D, isochronal (end of
900-ms pulse) current-voltage relations in the presence of increasing
external concentrations of BaCl
for wild-type and mutant
cIRK1. E, dose-response experiment at -150 mV. Points and bars represent the mean ± S.D. from
5 (wild-type) and 3 (Q125E) oocytes. Solid line represents the
best least-squares fit to: y = 1/(1 + (x/K)
), where K is the
value of x causing 50% inhibition. The best-fit parameters
were K = 11.73, n = 1.4 for wild-type;
and K = 2.1, n = 1.6 for Q125E. Dotted lines are curves drawn assuming the same values for K but n = 1.0.
We also studied the effect of Ba on cIRK1. It was
observed that cIRK1 currents appeared to be somewhat less sensitive to
block with Ba
than did those of the mouse inward
rectifier. For instance, IRK1 was completely blocked by 30 µM Ba
by 900 ms at both -130 and -160
mV(9) , while the same concentration of Ba
resulted in only a 70% average reduction in cIRK1 at -150
mV at 900 ms (the end of our pulse, Fig. 7). The ability of
Ba
to block current in Q125E was also determined. We
had observed that the sensitivity of cIRK1 to Ba
block was to some degree inversely proportional to the level of
expression; this effect was especially apparent for currents > 10
µA. (
)Thus, to study the effect of Ba
on wild-type and mutant channels, we studied oocytes expressing
currents within a range of 1-10 µA (at -150 mV).
Compared to the wild-type channel, Q125E had an increased sensitivity
to block with external Ba
(Fig. 7). The
best-fit parameters of a Langmuir isotherm were: K (WT)
= 12 µM, n
(WT) = 1.4;
and K (Q125E) = 2 µM, n
(Q125E) = 1.6. Although the apparent binding affinity K was increased about 6-fold, the Hill coefficients (n
) were similar.
Electrophysiological experiments have demonstrated an inward
rectifier potassium current to be present in apical hair
cells(8, 55) . It plays an important role in
determining the excitable properties of these cells, including the
resting membrane potential and the membrane time constant. We have
cloned an inward rectifier potassium channel from the chicken basilar
papilla. We have termed this channel cIRK1 for its close homology to
the mouse inward rectifier (IRK1). Two arguments would favor cIRK1
being the same as the inward rectifier potassium channel present in
tall hair cells. First, the electrophysiological properties of cIRK1
are similar to those of the inward rectifier channel present in apical
hair cells. In particular is its pronounced inward rectification, which
is an important feature of the inward rectifier channel in apical hair
cells. Second, we were able to demonstrate by RT-PCR that transcripts
encoding the channel were present in the apical half of the basilar
papilla, a result which parallels the electrophysiological data.
Although we obtained a PCR product whose sequence resembled the inward
rectifier ROMK-1, it is unlikely that this channel would be responsible
for the inward rectification in apical hair cells. This is because of
an inability to obtain a clone bearing this sequence on screening cDNA
libraries of 2 10
recombinants, and because ROMK-1
displays poor inward rectification(10) .
The limitation of the cIRK1 transcript to the apical half of the basilar papilla and the distribution of the calbindin transcript predominantly in the basal half of the basilar papilla would suggest that the differential distribution of these proteins is determined by control of transcription. This is direct evidence for such control within the inner ear epithelium. Should this process extend to other channels, as is suggested by the electrophysiological data, it would imply that a complex transcriptional regulatory process contributes to bringing about the tonotopicity in the basilar papilla.
The preferential
distribution of the calbindin transcript in the basal half of the
receptor epithelium also has implications to electrical tuning.
Electrical tuning is a mechanism by which the frequency of oscillation
in membrane potential defines the frequency of sound to which a given
hair cell best responds. It was first demonstrated in the
turtle(33) , and there is evidence that it also occurs in the
chick (8) , although some have questioned its significance in
the latter(34) . The graded oscillations in membrane potential
of hair cells across the epithelium are brought about by an interplay
between calcium-activated K channels and calcium
channels in individual hair cells (35) . The frequency of
oscillation in membrane potential of a given hair cell is determined by
the individual properties of the calcium-activated K
channel peculiar to that particular hair cell or by the calcium
buffering properties particular to that cell(36) . It has been
shown that the concentration of a cytoplasmic calcium buffer within
hair cells is sufficiently high to cause a spatial buffering of
Ca
within hair cells(37) . Calbindin is
thought to serve such a
function(32, 37, 38) . The differential
distribution of the calbindin transcript within the sensory epithelium
would suggest that an incremental Ca
buffering by
calbindin could contribute to determining the graded oscillation
frequencies and electrical tuning.
The six clones of 2.2 kb obtained on screening the cDNA library are unlikely to be full-length clones, even though they contained the entire open reading frame. This is suggested by the absence of a polyadenylation signal before the 3`-poly(A) end, and the major transcript of 5.4 kb present on Northern analysis of the different tissues including the cochlea. Furthermore, the transcripts encoding the analogous channels in the mouse (9) and the rat (16) are 5.4 kb in size. Since all the clones were of 2.2 kb in size, it would necessitate that the oligo(dT) primer primed at a site, rich in adenosines, internal to the poly(A) tail when the cDNA libraries were constructed. However, we cannot rule out the possibility that the cloned cDNA was derived from the 2.2-kb transcript detected on Northern analysis. The several less intense bands detected on Northern analysis in all these tissues would imply the existence of several other related mRNAs (channels) in these tissues.
All six clones obtained from the cDNA library had the same sequence and were not alternatively spliced. Furthermore, the gene for IRK1 in the mouse has been shown to be intronless within the open reading frame (39) . That there were no differences in the electrophysiological properties of the inward rectifier potassium currents in hair cells obtained from different locations in the apical basilar papilla is in keeping with these data(8) .
The
differences in the deduced amino acid sequence of cIRK1 compared to the
mouse channel (IRK1) were limited to two regions of the protein and may
have several implications. One region of difference was the short
sequence connecting the purported M1 and H5 regions. We identified a
single, polar but uncharged amino acid (Gln-125) lying within this
region of cIRK1 as being of importance in determining single-channel
conductance and sensitivity to Ba block. The
corresponding position in IRK1 contains a glutamate residue (which at
physiological pH would have a negative charge). cIRK1 had a reduced
single-channel conductance (17 pS using 150 mM external
K
) and reduced sensitivity to block with
Ba
compared to IRK1. IRK1 had a single-channel
conductance of 23 pS when using 140 mM external
K
(9) . Consistent with this observation, a
human homologue of IRK1, HIRK1(14) , which has a histidine in
this position, was found to have a single-channel conductance of 10 pS
as measured under somewhat different experimental conditions (100
mM external K
). IRK1 has been shown to have a
single-channel conductance of 21 pS when using 100 mM external
K
(19) . Changing glutamine 125 in cIRK1 to a
glutamate residue resulted in a channel which had a single-channel
conductance of 28 pS (using 150 mM external
K
). It also had an increased sensitivity to
Ba
block. The K value for Ba
block in the wild-type channel was 12 µM, while that
for Q125E was 2 µM. Although other inward rectifier
channels have been cloned that have different amino acids in this
position, it is difficult to compare these owing to the absence of
directly comparable electrophysiological data. Of particular interest
are the rat brain inward rectifier RB-IRK-2 (16) and the rabbit
cardiac channel RBHIK1 (15) which contain a glutamine and
glutamate, respectively, in this position.
The altered
single-channel conductance and sensitivity to Ba block in cIRK1 that was brought about by changing Gln-125 to a
glutamate lends itself to two explanations. First, it is possible that
Gln-125 lines the pore or the vestibule of the channel. Second, it is
also possible that the change in the electrophysiological properties of
the channel produced by this mutation are mediated by an allosteric
effect. Since two independent properties of the channel were changed in
a manner consistent with a direct interaction (see below), the first of
these possibilities is the more likely. This interpretation is also
consistent with the proposed membrane topology of the channel. Both
Gln-125 in cIRK1 and Glu-125 in IRK1 are preceded by a series of
charged amino acids (amino acids 112-117). A fortuitous change in
one of these amino acids between these two channels enables us to make
a prediction about its contribution to conductance and sensitivity to
Ba
block. A single glutamate residue (Glu-117) in
cIRK1 occupies the position of a lysine in IRK1. Such a reversal in
polarity would be predicted to increase the conductance and sensitivity
to external cation block in cIRK1 (see below). That it does not would
argue that this amino acid (Glu-117) does not contribute to determining
either of these properties.
The calculated Langmuir isotherms permit
us to deduce the importance of Gln-125 and by inference Glu-125 in
IRK1, to Ba binding. Consistent with a mechanism
involving multi-ion
block(40, 41, 42, 43) , we found a
Hill coefficient > 1. This would suggest that Ba
interacts at two (or more) sites. The mutant channel (Q125E) had
the same Hill coefficient, albeit with an increased sensitivity.
Although Gln-125 may form part of the Ba
binding site
within the pore, our results are also consistent with a hypothesis that
Glu-125 in IRK1 may lie in the vestibule of the pore and influence
conductance and Ba
block by controlling diffusion of
ions therein(44) . This is analogous to changes in conductance
mediated by negatively charged residues at the outer vestibule of the
nicotinic acetylcholine receptor channels(45) .
While
Asp-172 in IRK1 is important in determining inward rectification
mediated by internal Mg block(20, 21) , our results suggest that Glu-125
is an external residue that may play an important role in controlling
channel conductance. Both actions may involve electrostatic
interactions(46) . If K
channels are
tetrameric and exhibit 4-fold symmetry(47, 48) ,
Asp-172 and Glu-125 may form negatively charged rings at the inner and
outer mouths of the pore, respectively.
cIRK1 differed from IRK1
near the C terminus (amino acids 388-413). The C terminus has
been shown to confer some of the electrophysiological properties to the
inward rectifiers. Specifically, replacing the C terminus of ROMK-1
with that of IRK1 resulted in the chimeric channel having rectification
properties of IRK1 and a conductance intermediate between that of IRK1
and ROMK-1(19) . The electrophysiological properties of cIRK1
were, with the exception of conductance and sensitivity to external
Ba block, not measurably different from those
published properties of the mouse inward rectifier (IRK1). These two
properties that were different were shown to be mainly due to a single
amino acid change (Gln-125). Therefore, it could be reasoned that the
amino acids that are different at the C terminus are not critical to
determining these functional differences between the two channels.
Since rectification of the two channels was the same, an extension of
the same argument would suggest that these C-terminal amino acids that
are different are not critical, or not sufficiently different, to
affect this channel property.
At least three different potassium channels have been shown in electrophysiological experiments to be localized to different parts of the chick basilar papilla. Our results demonstrating the existence of cIRK1 transcripts within the apical basilar papilla would suggest that this channel, and, by implication, the other potassium channels within the basilar papilla are transcriptionally regulated. This would in turn necessitate complicated and minutely precise transcriptional regulatory mechanisms to bring about the differential expression of these and other proteins that determine tonotopicity within the 10,000 hair cells in the basilar papilla. Furthermore, our results relating to the alteration in conductance that is brought about by mutating Gln-125 has implications owing to a number of inward rectifier channels that differ in their conductances(49, 50, 51, 52) . This result suggests that one possible mechanism of altering conductance would be through changes in the external pore region. We would predict that this would be a site for natural mutations to occur thereby controlling conductance as physiologically required. This is particularly so since alteration of this residue does not seem to affect other channel properties such as its rate of activation and rectification.
This paper is dedicated to J. D. Priddle.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U20216[GenBank].